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Sample records for binding affibody molecules

  1. Generation of tumour-necrosis-factor-alpha-specific affibody molecules capable of blocking receptor binding in vitro.

    PubMed

    Jonsson, Andreas; Wållberg, Helena; Herne, Nina; Ståhl, Stefan; Frejd, Fredrik Y

    2009-08-17

    Affibody molecules specific for human TNF-alpha (tumour necrosis factor-alpha) were selected by phage-display technology from a library based on the 58-residue Protein A-derived Z domain. TNF-alpha is a proinflammatory cytokine involved in several inflammatory diseases and, to this day, four TNF-alpha-blocking protein pharmaceuticals have been approved for clinical use. The phage selection generated 18 unique cysteine-free affibody sequences of which 12 were chosen, after sequence cluster analysis, for characterization as proteins. Biosensor binding studies of the 12 Escherichia coli-produced and IMAC (immobilized-metal-ion affinity chromatography)-purified affibody molecules revealed three variants that demonstrated the strongest binding to human TNF-alpha. These three affibody molecules were subjected to kinetic binding analysis and also tested for their binding to mouse, rat and pig TNF-alpha. For ZTNF-alpha:185, subnanomolar affinity (KD=0.1-0.5 nM) for human TNF-alpha was demonstrated, as well as significant binding to TNF-alpha from the other species. Furthermore, the binding site was found to overlap with the binding site for the TNF-alpha receptor, since this interaction could be efficiently blocked by the ZTNF-alpha:185 affibody. When investigating six dimeric affibody constructs with different linker lengths, and one trimeric construct, it was found that the inhibition of the TNF-alpha binding to its receptor could be further improved by using dimers with extended linkers and/or a trimeric affibody construct. The potential implication of the results for the future design of affibody-based reagents for the diagnosis of inflammation is discussed.

  2. Evaluation of backbone-cyclized HER2-binding 2-helix affibody molecule for in vivo molecular imaging.

    PubMed

    Honarvar, Hadis; Jokilaakso, Nima; Andersson, Karl; Malmberg, Jennie; Rosik, Daniel; Orlova, Anna; Karlström, Amelie Eriksson; Tolmachev, Vladimir; Järver, Peter

    2013-04-01

    Affibody molecules, small scaffold proteins, have demonstrated an appreciable potential as imaging probes. Affibody molecules are composed of three alpha-helices. Helices 1 and 2 are involved in molecular recognition, while helix 3 provides stability. The size of Affibody molecules can be reduced by omitting the third alpha-helix and cross-linking the two remaining, providing a smaller molecule with better extravasation and quicker clearance of unbound tracer. The goal of this study was to develop a novel 2-helix Affibody molecule based on backbone cyclization by native chemical ligation (NCL). The HER2-targeting NCL-cyclized Affibody molecule ZHER2:342min has been designed, synthesized and site-specifically conjugated with a DOTA chelator. DOTA-ZHER2:342min was labeled with (111)In and (68)Ga. The binding affinity of DOTA-ZHER2:342min was evaluated in vitro. The targeting properties of (111)In- and (68)Ga-DOTA-ZHER2:342min were evaluated in mice bearing SKOV-3 xenografts and compared with the properties of (111)In- and (68)Ga-labeled PEP09239, a DOTA-conjugated 2-helix Affibody analogue cyclized by a homocysteine disulfide bridge. The dissociation constant (KD) for DOTA-ZHER2:342min binding to HER2 was 18nM according to SPR measurements. DOTA-ZHER2:342min was labeled with (111)In and (68)Ga. Both conjugates demonstrated bi-phasic binding kinetics to HER2-expressing cells, with KD1 in low nanomolar range. Both variants demonstrated specific uptake in HER2-expressing xenografts. Tumor-to-blood ratios at 2h p.i. were 6.1±1.3 for (111)In- DOTA-ZHER2:342min and 4.6±0.7 for (68)Ga-DOTA-ZHER2:342min. However, the uptake of DOTA-ZHER2:342min in lung, liver and spleen was appreciably higher than the uptake of PEP09239-based counterparts. Native chemical ligation enables production of a backbone-cyclized HER2-binding 2-helix Affibody molecule (ZHER2:342min) with low nanomolar target affinity and specific tumor uptake. Copyright © 2013 Elsevier Inc. All rights reserved.

  3. A new prodrug form of Affibody molecules (pro-Affibody) is selectively activated by cancer-associated proteases.

    PubMed

    Sandersjöö, Lisa; Jonsson, Andreas; Löfblom, John

    2015-04-01

    Affinity proteins have advanced the field of targeted therapeutics due to their generally higher specificity compared to small molecular compounds. However, side effects caused by on-target binding in healthy tissues are still an issue. Here, we design and investigate a prodrug strategy for improving tissue specificity of Affibody molecules in future in vivo studies. The prodrug Affibody (pro-Affibody) against the HER2 receptor was constructed by fusing a HER2-specific Affibody (ZHER2) to an anti-idiotypic Affibody (anti-ZHER2). The linker was engineered to comprise a substrate peptide for the cancer-associated matrix metalloprotease 1 (MMP-1). The hypothesis was that the binding surface of ZHER2 would thereby be blocked from interacting with HER2 until the substrate peptide was specifically hydrolyzed by MMP-1. Binding should thereby only occur where MMP-1 is overexpressed, potentially decreasing on-target toxicities in normal tissues. The pro-Affibody was engineered to find a suitable linker and substrate peptide, and the different constructs were evaluated with a new bacterial display assay. HER2-binding of the pro-Affibody was efficiently masked and proteolytic activation of the best variant yielded over 1,000-fold increase in apparent binding affinity. Biosensor analysis revealed that blocking of the pro-Affibody primarily affected the association phase. In a cell-binding assay, the activated pro-Affibody targeted native HER2 on cancer cells as opposed to the non-activated pro-Affibody. We believe this prodrug approach with proteolytic activation is promising for improving tissue specificity in future in vivo targeting applications and can hopefully be extended to other Affibody molecules and similar affinity proteins as well.

  4. Affibody molecules as engineered protein drugs

    PubMed Central

    Frejd, Fredrik Y; Kim, Kyu-Tae

    2017-01-01

    Affibody molecules can be used as tools for molecular recognition in diagnostic and therapeutic applications. There are several preclinical studies reported on diagnostic and therapeutic use of this molecular class of alternative scaffolds, and early clinical evidence is now beginning to accumulate that suggests the Affibody molecules to be efficacious and safe in man. The small size and ease of engineering make Affibody molecules suitable for use in multispecific constructs where AffiMabs is one such that offers the option to potentiate antibodies for use in complex disease. PMID:28336959

  5. Engineering of a bispecific affibody molecule towards HER2 and HER3 by addition of an albumin-binding domain allows for affinity purification and in vivo half-life extension.

    PubMed

    Malm, Magdalena; Bass, Tarek; Gudmundsdotter, Lindvi; Lord, Martin; Frejd, Fredrik Y; Ståhl, Stefan; Löfblom, John

    2014-09-01

    Emerging strategies in cancer biotherapy include the generation and application of bispecific antibodies, targeting two tumor-associated antigens for improved tumor selectivity and potency. Here, an alternative format for bispecific molecules was designed and investigated, in which two Affibody molecules were linked by an albumin-binding domain (ABD). Affibody molecules are small (6 kDa) affinity proteins and this new format allows for engineering of molecules with similar function as full-length bispecific antibodies, but in a dramatically smaller size (around eight-fold smaller). The ABD was intended to function both as a tag for affinity purification as well as for in vivo half-life extension in future preclinical and clinical investigations. Affinity-purified bispecific Affibody molecules, targeting HER2 and HER3, showed simultaneous binding to the three target proteins (HER2, HER3, and albumin) when investigated in biosensor assays. Moreover, simultaneous interactions with the receptors and albumin were demonstrated using flow cytometry on cancer cells. The bispecific Affibody molecules were also able to block ligand-induced phosphorylation of the HER receptors, indicating an anti-proliferative effect. We believe that this compact and flexible format has great potential for developing new potent bispecific affinity proteins in the future, as it combines the benefits of a small size (e.g. improved tissue penetration and reduced cost of goods) with a long circulatory half-life.

  6. In vivo and in vitro studies on renal uptake of radiolabeled affibody molecules for imaging of HER2 expression in tumors.

    PubMed

    Altai, Mohamed; Varasteh, Zohreh; Andersson, Karl; Eek, Annemarie; Boerman, Otto; Orlova, Anna

    2013-04-01

    Affibody molecules (6-7 kDa) are a new class of small robust three-helical scaffold proteins. Radiolabeled subnanomolar anti-HER2 affibody ZHER2:342 was developed for imaging of HER2 expression in tumors, and a clinical study has demonstrated that the (111)In- and (68)Ga-labeled affibody molecules can efficiently detect HER2 expressing metastases in breast cancer patients. However, a significant renal accumulation of radioactivity after systemic injection of a radiolabeled anti-HER2 affibody conjugate is observed. The aim of this study was to investigate the mechanism of renal reabsorption of anti-HER2 affibody at the molecular level. Renal accumulation of radiolabeled anti-HER2 affibody molecules was studied in a murine model and in vitro using opossum-derived proximal tubule (OK) cells. It was found that kidney reabsorption of affibody molecule was not driven by megalin/cubilin. Amino acids in the target-binding side of affibody molecule were involved in binding to OK cells. On OK cells, two types of receptors for anti-HER2 affibody molecule were found: KD1=0.8 nM, Bmax1=71,500 and KD2=9.2 nM, Bmax2=367,000. The results of the present study indicate that affibody molecule and other scaffold-based targeting proteins with a relatively low kidney uptake can be selected using in vitro studies with tubular kidney cells.

  7. Generation of Affibody ligands binding interleukin-2 receptor alpha/CD25.

    PubMed

    Grönwall, Caroline; Snelders, Eveline; Palm, Anna Jarelöv; Eriksson, Fredrik; Herne, Nina; Ståhl, Stefan

    2008-06-01

    Affibody molecules specific for human IL-2Ralpha, the IL-2 (interleukin-2) receptor alpha subunit, also known as CD25, were selected by phage-display technology from a combinatorial protein library based on the 58-residue Protein A-derived Z domain. The IL-2R system plays a major role in T-cell activation and the regulation of cellular immune responses. Moreover, CD25 has been found to be overexpressed in organ rejections, a number of autoimmune diseases and T-cell malignancies. The phage-display selection using Fc-fused target protein generated 16 unique Affibody molecules targeting CD25. The two most promising binders were characterized in more detail using biosensor analysis and demonstrated strong and selective binding to CD25. Kinetic biosensor analysis revealed that the two monomeric Affibody molecules bound to CD25 with apparent affinities of 130 and 240 nM respectively. The Affibody molecules were, on biosensor analysis, found to compete for the same binding site as the natural ligand IL-2 and the IL-2 blocking monoclonal antibody 2A3. Hence the Affibody molecules were assumed to have an overlapping binding site with IL-2 and antibodies targeting the IL-2 blocking Tac epitope (for example, the monoclonal antibodies Daclizumab and Basiliximab, both of which have been approved for therapeutic use). Furthermore, immunofluorescence microscopy and flow-cytometric analysis of CD25-expressing cells demonstrated that the selected Affibody molecules bound to CD4+ CD25+ PMBCs (peripheral-blood mononuclear cells), the IL-2-dependent cell line NK92 and phytohaemagglutinin-activated PMBCs. The potential use of the CD25-binding Affibody molecules as targeting agents for medical imaging and for therapeutic applications is discussed.

  8. Generation of affibody molecules specific for HPV16 E7 recognition

    PubMed Central

    Zhang, Chanqiong; Song, Yiling; Cai, Yiqi; Cen, Danwei; Wang, Ledan; Xiong, Yirong; Jiang, Pengfei; Zhu, Shanli; Zhao, Kong-Nan; Zhang, Lifang

    2016-01-01

    Cervical cancer caused by infection with high-risk human papillomavirus remains to be the most deadly gynecologic malignancy worldwide. It is well documented that persistent expression of two oncogenes (E6/E7) plays the key roles in cervical cancer. Thus, in vivo detection of the oncoproteins is very important for the diagnosis of the cancer. Recently, affibody molecules have been demonstrated to be a powerful targeting probe for tumor–targeted imaging and diagnosis. In this study, four HPV16 E7-binding affibody molecules (ZHPV16 E7127, ZHPV16E7301, ZHPV16E7384 and ZHPV16E7745) were screened from a phage-displayed peptide library and used for molecular imaging in tumor-bearing mice. Biosensor binding analyses showed first that the four affibody molecules bound to HPV16 E7 with very high affinity and specificity. They co-localized with E7 protein only in two HPV16-positive cancer cells (SiHa and CaSki). Furthermore, affibody ZHPV16E7384 was conjugated with Dylight755 and used for in vivo tumor-imaging. Strongly high-contrast tumor retention of this affibody only occurred in HPV16-derived tumors of mice as early as 30 min post-injection, not in HPV-negative and HPV18-derived tumors. The accumulation of Dylight755-conjugated ZHPV16E7384 in tumor was achieved over a longer time period (24 h). The data here provide strong evidence that E7-specific affibody molecules have great potential used for molecular imaging and diagnosis of HPV-induced cancers. PMID:27659535

  9. Affibody molecules for in vivo characterization of HER2-positive tumors by near-infrared imaging.

    PubMed

    Lee, Sang Bong; Hassan, Moinuddin; Fisher, Robert; Chertov, Oleg; Chernomordik, Victor; Kramer-Marek, Gabriela; Gandjbakhche, Amir; Capala, Jacek

    2008-06-15

    HER2 overexpression has been associated with a poor prognosis and resistance to therapy in breast cancer patients. We are developing molecular probes for in vivo quantitative imaging of HER2 receptors using near-infrared (NIR) optical imaging. The goal is to provide probes that will minimally interfere with the studied system, that is, whose binding does not interfere with the binding of the therapeutic agents and whose effect on the target cells is minimal. We used three different types of HER2-specific Affibody molecules [monomer ZHER2:342, dimer (ZHER2:477)2, and albumin-binding domain-fused-(ZHER2:342)2] as targeting agents and labeled them with Alexa Fluor dyes. Trastuzumab was also conjugated, using commercially available kits, as a standard control. The resulting conjugates were characterized in vitro by toxicity assays, Biacore affinity measurements, flow cytometry, and confocal microscopy. Semiquantitative in vivo NIR optical imaging studies were carried out using mice with s.c. xenografts of HER2-positive tumors. The HER2-specific Affibody molecules were not toxic to HER2-overexpressing cells and their binding to HER2 did interfere with neither binding nor effectives of trastuzumab. The binding affinities and specificities of the Affibody-Alexa Fluor fluorescent conjugates to HER2 were unchanged or minimally affected by the modifications. Pharmacokinetics and biodistribution studies showed the albumin-binding domain-fused-(ZHER2:342)2-Alexa Fluor 750 conjugate to be an optimal probe for optical imaging of HER2 in vivo. Our results suggest that Affibody-Alexa Fluor conjugates may be used as a specific NIR probe for the noninvasive semiquantitative imaging of HER2 expression in vivo.

  10. Evaluation of a maleimido derivative of CHX-A” DTPA for site-specific labeling of Affibody molecules

    PubMed Central

    Tolmachev, Vladimir; Xu, Heng; Wållberg, Helena; Ahlgren, Sara; Hjertman, Magnus; Sjöberg, Anna; Sandström, Mattias; Abrahmsén, Lars; Brechbiel, Martin W.; Orlova, Anna

    2008-01-01

    Affibody molecules are a new class of small targeting proteins based on a common threehelix bundle structure. Affibody molecules binding a desired target may be selected using phage-display technology. An Affibody molecule ZHER2:342 binding with subnanomolar affinity to the tumor antigen HER2 has recently been developed for radionuclide imaging in vivo. Introduction of a single cysteine into the cysteine-free Affibody scaffold provides a unique thiol group for site-specific labeling of recombinant Affibody molecules. The recently developed maleimido-CHX-A” DTPA was site-specifically conjugated at the C-terminal cysteine of ZHER2:2395-C, a variant of ZHER2:342, providing a homogenous conjugate with a dissociation constant of 56 pM. The yield of labeling with 111In was > 99% after 10 min at room temperature. In vitro cell tests demonstrated specific binding of 111In-CHX-A” DTPAZ2395-C to HER2-expressing cell-line SKOV-3 and good cellular retention of radioactivity. In normal mice, the conjugate demonstrated rapid clearance from all non-specific organs except kidney. In mice bearing SKOV-3 xenografts, the tumor uptake of 111In-CHX-A” DTPAZ2395-C was 17.3 ± 4.8 % IA/g and the tumor-to-blood ratio 86 ± 46 (4 h post-injection). HER2-exprssing xenografts were clearly visualized 1 h post-injection. In conclusion, coupling of maleimido-CHX-A” DTPA to cysteine-containing Affibody molecules provides welldefined uniform conjugate, which can be rapidly labeled at room temperature and provides high-contrast imaging of molecular targets in vivo. PMID:18620447

  11. Evaluation of a maleimido derivative of CHX-A'' DTPA for site-specific labeling of affibody molecules.

    PubMed

    Tolmachev, Vladimir; Xu, Heng; Wållberg, Helena; Ahlgren, Sara; Hjertman, Magnus; Sjöberg, Anna; Sandström, Mattias; Abrahmsén, Lars; Brechbiel, Martin W; Orlova, Anna

    2008-08-01

    Affibody molecules are a new class of small targeting proteins based on a common three-helix bundle structure. Affibody molecules binding a desired target may be selected using phage-display technology. An Affibody molecule Z HER2:342 binding with subnanomolar affinity to the tumor antigen HER2 has recently been developed for radionuclide imaging in vivo. Introduction of a single cysteine into the cysteine-free Affibody scaffold provides a unique thiol group for site-specific labeling of recombinant Affibody molecules. The recently developed maleimido-CHX-A'' DTPA was site-specifically conjugated at the C-terminal cysteine of Z HER2:2395-C, a variant of Z HER2:342, providing a homogeneous conjugate with a dissociation constant of 56 pM. The yield of labeling with (111)In was >99% after 10 min at room temperature. In vitro cell tests demonstrated specific binding of (111)In-CHX-A'' DTPA-Z 2395-C to HER2-expressing cell-line SKOV-3 and good cellular retention of radioactivity. In normal mice, the conjugate demonstrated rapid clearance from all nonspecific organs except kidney. In mice bearing SKOV-3 xenografts, the tumor uptake of (111)In-CHX-A'' DTPA-Z 2395-C was 17.3 +/- 4.8% IA/g and the tumor-to-blood ratio 86 +/- 46 (4 h postinjection). HER2-expressing xenografts were clearly visualized 1 h postinjection. In conclusion, coupling of maleimido-CHX-A'' DTPA to cysteine-containing Affibody molecules provides a well-defined uniform conjugate, which can be rapidly labeled at room temperature and provides high-contrast imaging of molecular targets in vivo.

  12. Feasibility of Affibody Molecule-Based PNA-Mediated Radionuclide Pretargeting of Malignant Tumors

    PubMed Central

    Honarvar, Hadis; Westerlund, Kristina; Altai, Mohamed; Sandström, Mattias; Orlova, Anna; Tolmachev, Vladimir; Karlström, Amelie Eriksson

    2016-01-01

    Affibody molecules are small (7 kDa), non-immunoglobulin scaffold proteins with a potential as targeting agents for radionuclide imaging of cancer. However, high renal re-absorption of Affibody molecules prevents their use for radionuclide therapy with residualizing radiometals. We hypothesized that the use of Affibody-based peptide nucleic acid (PNA)-mediated pretargeting would enable higher accumulation of radiometals in tumors than in kidneys. To test this hypothesis, we designed an Affibody-PNA chimera ZHER2:342-SR-HP1 containing a 15-mer HP1 PNA recognition tag and a complementary HP2 hybridization probe permitting labeling with both 125I and 111In. 111In-ZHER2:342-SR-HP1 bound specifically to HER2-expressing BT474 and SKOV-3 cancer cells in vitro, with a KD of 6±2 pM for binding to SKOV-3 cells. Specific high affinity binding of the radiolabeled complementary PNA probe 111In-/125I-HP2 to ZHER2:342-SR-HP1 pre-treated cells was demonstrated. 111In-ZHER2:342-SR-HP1 demonstrated specific accumulation in SKOV-3 xenografts in BALB/C nu/nu mice and rapid clearance from blood. Pre-saturation of SKOV-3 with non-labeled anti-HER2 Affibody or the use of HER2-negative Ramos xenografts resulted in significantly lower tumor uptake of 111In-ZHER2:342-SR-HP1. The complementary PNA probe 111In/125I-HP2 accumulated in SKOV-3 xenografts when ZHER2:342-SR-HP1 was injected 4 h earlier. The tumor accumulation of 111In/125I-HP2 was negligible without ZHER2:342-SR-HP1 pre-injection. The uptake of 111In-HP2 in SKOV-3 xenografts was 19±2 %ID/g at 1 h after injection. The uptake in blood and kidneys was approximately 50- and 2-fold lower, respectively. In conclusion, we have shown that the use of Affibody-based PNA-mediated pretargeting enables specific delivery of radiometals to tumors and provides higher radiometal concentration in tumors than in kidneys. PMID:26722376

  13. Human Serum Albumin and HER2-Binding Affibody Fusion Proteins for Targeted Delivery of Fatty Acid-Modified Molecules and Therapy.

    PubMed

    Dong, Daoyuan; Xia, Guanjun; Li, Zhijun; Li, Zhiyu

    2016-10-03

    Human epidermal growth factor receptor 2 (HER2) is a well-studied therapeutic target as well as a biomarker of breast cancer. HER2-targeting affibody (ZHER2:342) is a novel small scaffold protein with an extreme high affinity against HER2 screened by phage display. However, the small molecular weight of ZHER2:342 has limited its pharmaceutical application. Human serum albumin (HSA) and ZHER2:342 fusion protein may not only extend the serum half-life of ZHER2:342 but also preserve the biological function of HSA to bind and transport fatty acids, which can be used to deliver fatty acid-modified therapeutics to HER2-positive cancer cells. Two HSA and ZHER2:342 fusion proteins, one with a single ZHER2:342 domain fused to the C terminus of HSA (rHSA-ZHER2) and another with two tandem copies of ZHER2:342 fused to the C terminus of HSA (rHSA-(ZHER2)2), have been constructed, expressed, and purified. Both fusion proteins possessed the HER2 and fatty acid (FA) binding abilities demonstrated by in vitro assays. Interestingly, rHSA-(ZHER2)2, not rHSA-ZHER2, was able to inhibit the proliferation of SK-BR-3 cells at a relatively low concentration, and the increase of HER2 and ERK1/2 phosphorylation followed by rHSA-(ZHER2)2 treatment has been observed. HSA fusion proteins are easy and economical to express, purify, and formulate. As expected, HSA fusion proteins and fusion protein-bound fatty acid-modified FITC could be efficiently taken up by cells. These results proved the feasibility of using HSA fusion proteins as therapeutic agents as well as carriers for targeted drug delivery.

  14. Molecular design and optimization of 99mTc-labeled recombinant affibody molecules improves their biodistribution and imaging properties.

    PubMed

    Wållberg, Helena; Orlova, Anna; Altai, Mohammed; Hosseinimehr, Seyed Jalal; Widström, Charles; Malmberg, Jennie; Ståhl, Stefan; Tolmachev, Vladimir

    2011-03-01

    Affibody molecules are a recently developed class of targeting proteins based on a nonimmunoglobulin scaffold. The small size (7 kDa) and subnanomolar affinity of Affibody molecules enables high-contrast imaging of tumor-associated molecular targets, particularly human epidermal growth factor receptor type 2 (HER2). (99m)Tc as a label offers advantages in clinical practice, and earlier studies demonstrated that (99m)Tc-labeled recombinant Affibody molecules with a C-terminal cysteine could be used for HER2 imaging. However, the renal retention of radioactivity exceeded tumor uptake, which might complicate imaging of metastases in the lumbar region. The aim of this study was to develop an agent with low renal uptake and preserved tumor targeting. A series of recombinant derivatives of the HER2-binding Z(HER2)(:342) Affibody molecule with a C-terminal chelating sequence, -GXXC (X denoting glycine, serine, lysine, or glutamate), was designed. The constructs were labeled with (99m)Tc and evaluated in vitro and in vivo. All variants were stably labeled with (99m)Tc, with preserved capacity to bind specifically to HER2-expressing cells in vitro and in vivo. The composition of the chelating sequence had a clear influence on the cellular processing and biodistribution properties of the Affibody molecules. The best variant, (99m)Tc-Z(HER2)(:V2), with the C-terminal chelating sequence -GGGC, provided the lowest radioactivity retention in all normal organs and tissues including the kidneys. (99m)Tc-Z(HER2)(:V2) displayed high uptake of radioactivity in HER2-expressing xenografts, 22.6 ± 4.0 and 7.7 ± 1.5 percentage injected activity per gram of tissue at 4 h after injection in SKOV-3 (high HER2 expression) and DU-145 (low HER2 expression) tumors, respectively. In both models, the tumor uptake exceeded the renal uptake. These results demonstrate that the biodistribution properties of recombinant (99m)Tc-labeled Affibody molecules can be optimized by modification of the C

  15. Influence of nuclides and chelators on imaging using affibody molecules: comparative evaluation of recombinant affibody molecules site-specifically labeled with ⁶⁸Ga and ¹¹¹In via maleimido derivatives of DOTA and NODAGA.

    PubMed

    Altai, Mohamed; Strand, Joanna; Rosik, Daniel; Selvaraju, Ram Kumar; Eriksson Karlström, Amelie; Orlova, Anna; Tolmachev, Vladimir

    2013-06-19

    Accurate detection of cancer-associated molecular abnormalities in tumors could make cancer treatment more personalized. Affibody molecules enable high contrast imaging of tumor-associated protein expression shortly after injection. The use of the generator-produced positron-emitting radionuclide (68)Ga should increase sensitivity of HER2 imaging. The chemical nature of radionuclides and chelators influences the biodistribution of Affibody molecules, providing an opportunity to further increase the imaging contrast. The aim of the study was to compare maleimido derivatives of DOTA and NODAGA for site-specific labeling of a recombinant ZHER2:2395 HER2-binding Affibody molecule with (68)Ga. DOTA and NODAGA were site-specifically conjugated to the ZHER2:2395 Affibody molecule having a C-terminal cysteine and labeled with (68)Ga and (111)In. All labeled conjugates retained specificity to HER2 in vitro. Most of the cell-associated activity was membrane-bound with a minor difference in internalization rate. All variants demonstrated specific targeting of xenografts and a high tumor uptake. The xenografts were clearly visualized using all conjugates. The influence of chelator on the biodistribution and targeting properties was much less pronounced for (68)Ga than for (111)In. The tumor uptake of (68)Ga-NODAGA-ZHER2:2395 and (68)Ga-DOTA-ZHER2:2395 and tumor-to-blood ratios at 2 h p.i. did not differ significantly. However, the tumor-to-liver ratio was significantly higher for (68)Ga-NODAGA- ZHER2:2395 (8 ± 2 vs 5.0 ± 0.3) offering the advantage of better liver metastases visualization. In conclusion, influence of chelators on biodistribution of Affibody molecules depends on the radionuclides and reoptimization of labeling chemistry is required when a radionuclide label is changed.

  16. Affibody molecules: potential for in vivo imaging of molecular targets for cancer therapy.

    PubMed

    Tolmachev, Vladimir; Orlova, Anna; Nilsson, Fredrik Y; Feldwisch, Joachim; Wennborg, Anders; Abrahmsén, Lars

    2007-04-01

    Targeting radionuclide imaging of tumor-associated antigens may help to select patients who will benefit from a particular biological therapy. Affibody molecules are a novel class of small (approximately 7 kDa) phage display-selected affinity proteins, based on the B-domain scaffold of staphylococcal protein A. A large library (3 x 10(9) variants) has enabled selection of high-affinity (up to 22 pM) binders for a variety of tumor-associated antigens. The small size of Affibody molecules provides rapid tumor localization and fast clearance from nonspecific compartments. Preclinical studies have demonstrated the potential of Affibody molecules for specific and high-contrast radionuclide imaging of HER2 in vivo, and pilot clinical data using indium-111 and gallium-68 labeled anti-HER2 Affibody tracer have confirmed its utility for radionuclide imaging in cancer patients.

  17. Evaluation of a maleimido derivative of NOTA for site-specific labeling of affibody molecules.

    PubMed

    Tolmachev, Vladimir; Altai, Mohamed; Sandström, Mattias; Perols, Anna; Karlström, Amelie Eriksson; Boschetti, Frederic; Orlova, Anna

    2011-05-18

    Radionuclide molecular imaging has the potential to improve cancer treatment by selection of patients for targeted therapy. Affibody molecules are a class of small (7 kDa) high-affinity targeting proteins with appreciable potential as molecular imaging probes. The NOTA chelator forms stable complexes with a number of radionuclides suitable for SPECT or PET imaging. A maleimidoethylmonoamide NOTA (MMA-NOTA) has been prepared for site-specific labeling of Affibody molecules having a unique C-terminal cysteine. Coupling of the MMA-NOTA to the anti-HER2 Affibody molecule Z(HER2:2395) resulted in a conjugate with an affinity (dissociation constant) to HER2 of 72 pM. Labeling of [MMA-NOTA-Cys(61)]-Z(HER2:2395) with (111)In gave a yield of >95% after 20 min at 60 °C. In vitro cell tests demonstrated specific binding of [(111)In-MMA-NOTA-Cys(61)]-Z(HER2:2395) to HER2-expressing cell lines. In mice bearing prostate cancer DU-145 xenografts, the tumor uptake of [(111)In-MMA-NOTA-Cys(61)]-Z(HER2:2395) was 8.2 ± 0.9% IA/g and the tumor-to-blood ratio was 31 ± 1 (4 h postinjection). DU-145 xenografts were clearly visualized by a gamma camera. Direct in vivo comparison of [(111)In-MMA-NOTA-Cys(61)]-Z(HER2:2395) and [(111)In-MMA-DOTA-Cys(61)]-Z(HER2:2395) demonstrated that both conjugates provided equal radioactivity uptake in tumors, but the tumor-to-organ ratios were better for [(111)In-MMA-NOTA-Cys(61)]-Z(HER2:2395) due to more efficient clearance from normal tissues. In conclusion, coupling of MMA-NOTA to a cysteine-containing Affibody molecule resulted in a site-specifically labeled conjugate, which retains high affinity, can be efficiently labeled, and allows for high-contrast imaging.

  18. Synthesis and chemoselective intramolecular crosslinking of a HER2-binding affibody.

    PubMed

    Ekblad, Torun; Tolmachev, Vladimir; Orlova, Anna; Lendel, Christofer; Abrahmsén, Lars; Karlström, Amelie Eriksson

    2009-01-01

    The human epidermal growth factor receptor HER2 has emerged as an important target for molecular imaging of breast cancer. This article presents the design and synthesis of a HER2-targeting affibody molecule with improved stability and tumor targeting capacity, and with potential use as an imaging agent. The 58 aa three-helix bundle protein was assembled using solid-phase peptide synthesis, and a chemoselective ligation strategy was used to establish an intramolecular thioether bond between the side chain thiol group of a cysteine residue, positioned in the loop between helices I and II, and a chloroacetyl group on the side chain amino group of the C-terminal lysine residue. The tethered protein offered an increased thermal stability, with a melting temperature of 64 degrees C, compared to 54 degrees C for the linear control. The ligation did not have a major influence on the HER2 binding affinity, which was 320 and 380 pM for the crosslinked and linear molecules, respectively. Biodistribution studies were performed both in normal and tumor-bearing mice to evaluate the impact of the crosslinking on the in vivo behavior and on the tumor targeting performance. The distribution pattern was characterized by a low uptake in all organs except kidney, and rapid clearance from blood and normal tissue. Crosslinking of the protein resulted in a significantly increased tumor accumulation, rendering the tethered HER2-binding affibody molecule a valuable lead in the development of superior HER2 imaging agents.

  19. Improved tumor-to-organ ratios of a novel 67Ga-human epidermal growth factor radionuclide conjugate with preadministered antiepidermal growth factor receptor affibody molecules.

    PubMed

    Sandström, Karl; Haylock, Anna-Karin; Velikyan, Irina; Spiegelberg, Diana; Kareem, Heewa; Tolmachev, Vladimir; Lundqvist, Hans; Nestor, Marika

    2011-10-01

    The overexpression of the epidermal growth factor receptor (EGFR) in head and neck squamous cell carcinoma (HNSCC) is associated with poor prognosis. Targeted nuclear imaging of the EGFR expression could improve the diagnostics in patients with HNSCC. However, the high expression of EGFR in normal organs may conceal the tumor uptake and therefore limit the use. This study assesses the biodistribution of a novel human epidermal growth factor (hEGF) radionuclide conjugate after preinjection with anti-EGFR affibody molecules. hEGF was conjugated with p-SCN-Bn-NOTA and labeled with (67)Ga. The biodistribution of [(67)Ga]Ga-NOTA-Bn-NCS-hEGF in nude mice with EGFR-expressing xenografts was evaluated either alone or 45 minutes after preinjection with one of the anti-EGFR affibody molecules Z(EGFR:1907), (Z(EGFR:1907))(2), or (Z(EGFR:955))(2). The novel radioimmunoconjugate, [(67)Ga]Ga-NOTA-Bn-NCS-hEGF, demonstrated high stability in vitro and specific binding to hEGF in vitro and in vivo. Preinjection with anti-EGFR affibody molecules improved the tumor-to-organ ratio in the liver, salivary glands, and colon. Overall, the dimeric high-affinity affibody molecule (Z(EGFR:1907))(2) exhibited the best results. These findings show that preblocking with an anti-EGFR affibody molecule is a promising tool that could improve the outcome of radionuclide-based imaging of EGFR-expressing tumors.

  20. Imaging of CAIX-expressing xenografts in vivo using 99mTc-HEHEHE-ZCAIX:1 Affibody molecule

    PubMed Central

    HONARVAR, HADIS; GAROUSI, JAVAD; GUNNERIUSSON, ELIN; HÖIDÉN-GUTHENBERG, INGMARIE; ALTAI, MOHAMED; WIDSTRÖM, CHARLES; TOLMACHEV, VLADIMIR; FREJD, FREDRIK Y.

    2015-01-01

    Carbonic anhydrase IX (CAIX) is a transmembrane enzyme involved in regulation of tissue pH balance. In cancer, CAIX expression is associated with tumor hypoxia. CAIX is also overexpressed in renal cell carcinoma and is a molecular target for the therapeutic antibody cG250 (girentuximab). Radionuclide imaging of CAIX expression might be used for identification of patients who may benefit from cG250 therapy and from treatment strategies for hypoxic tumors. Affibody molecules are small (7 kDa) scaffold proteins having a high potential as probes for radionuclide molecular imaging. The aim of the present study was to evaluate feasibility of in vivo imaging of CAIX-expression using radiolabeled Affibody molecules. A histidine-glutamate-histidine-glutamate-histidine-glutamate (HE)3-tag-containing CAIX-binding Affibody molecule (HE)3-ZCAIX:1 was labeled with [99mTc(CO)3]+. Its binding properties were evaluated in vitro using CAIX-expressing SK-RC-52 renal carcinoma cells. 99mTc-(HE)3-ZCAIX:1 was evaluated in NMRI nu/nu mice bearing SK-RC-52 xenografts. The in vivo specificity test confirmed CAIX-mediated tumor targeting. 99mTc-(HE)3-ZCAIX:1 cleared rapidly from blood and normal tissues except for kidneys. At optimal time-point (4 h p.i.), the tumor uptake was 9.7±0.7% ID/g, and tumor-to-blood ratio was 53±10. Experimental imaging of CAIX-expressing SK-RC-52 xenografts at 4 h p.i. provided high contrast images. The use of radioiodine label for ZCAIX:1 enabled the reduction of renal uptake, but resulted in significantly lower tumor uptake and tumor-to-blood ratio. Results of the present study suggest that radiolabeled Affibody molecules are promising probes for imaging of CAIX-expression in vivo. PMID:25434612

  1. Design, Preparation, and Characterization of PNA-Based Hybridization Probes for Affibody-Molecule-Mediated Pretargeting.

    PubMed

    Westerlund, Kristina; Honarvar, Hadis; Tolmachev, Vladimir; Eriksson Karlström, Amelie

    2015-08-19

    In radioimmunotherapy, the contrast between tumor and normal tissue can be improved by using a pretargeting strategy with a primary targeting agent, which is conjugated to a recognition tag, and a secondary radiolabeled molecule binding specifically to the recognition tag. The secondary molecule is injected after the targeting agent has accumulated in the tumor and is designed to have a favorable biodistribution profile, with fast clearance from blood and low uptake in normal tissues. In this study, we have designed and evaluated two complementary peptide nucleic acid (PNA)-based probes for specific and high-affinity association in vivo. An anti-HER2 Affibody-PNA chimera, Z(HER2:342)-SR-HP1, was produced by a semisynthetic approach using sortase A catalyzed ligation of a recombinantly produced Affibody molecule to a PNA-based HP1-probe assembled using solid-phase chemistry. A complementary HP2 probe carrying a DOTA chelator and a tyrosine for dual radiolabeling was prepared by solid-phase synthesis. Circular dichroism (CD) spectroscopy and UV thermal melts showed that the probes can hybridize to form a structured duplex with a very high melting temperature (T(m)), both in HP1:HP2 and in Z(HER2:342)-SR-HP1:HP2 (T(m) = 86-88 °C), and the high binding affinity between Z(HER2:342)-SR-HP1 and HP2 was confirmed in a surface plasmon resonance (SPR)-based binding study. Following a moderately fast association (1.7 × 10(5) M(-1) s(-1)), the dissociation of the probes was extremely slow and <5% dissociation was observed after 17 h. The equilibrium dissociation constant (K(D)) for Z(HER2:342)-SR-HP1:HP2 binding to HER2 was estimated by SPR to be 212 pM, suggesting that the conjugation to PNA does not impair Affibody binding to HER2. The biodistribution profiles of (111)In- and (125)I-labeled HP2 were measured in NMRI mice, showing very fast blood clearance rates and low accumulation of radioactivity in kidneys and other organs. The measured radioactivity in blood was 0.63

  2. Comparative Evaluation of Affibody Molecules for Radionuclide Imaging of in Vivo Expression of Carbonic Anhydrase IX.

    PubMed

    Garousi, Javad; Honarvar, Hadis; Andersson, Ken G; Mitran, Bogdan; Orlova, Anna; Buijs, Jos; Löfblom, John; Frejd, Fredrik Y; Tolmachev, Vladimir

    2016-11-07

    Overexpression of the enzyme carbonic anhydrase IX (CAIX) is documented for chronically hypoxic malignant tumors as well as for normoxic renal cell carcinoma. Radionuclide molecular imaging of CAIX would be useful for detection of hypoxic areas in malignant tumors, for patients' stratification for CAIX-targeted therapies, and for discrimination of primary malignant and benign renal tumors. Earlier, we have reported feasibility of in vivo radionuclide based imaging of CAIX expressing tumors using Affibody molecules, small affinity proteins based on a nonimmunoglobulin scaffold. In this study, we compared imaging properties of several anti-CAIX Affibody molecules having identical scaffold parts and competing for the same epitope on CAIX, but having different binding paratopes. Four variants were labeled using residualizing (99m)Tc and nonresidualizing (125)I labels. All radiolabeled variants demonstrated high-affinity detection of CAIX-expressing cell line SK-RC-52 in vitro and specific accumulation in SK-RC-52 xenografts in vivo. (125)I-labeled conjugates demonstrated much lower radioactivity uptake in kidneys but higher radioactivity concentration in blood compared with (99m)Tc-labeled counterparts. Although all variants cleared rapidly from blood and nonspecific compartments, there was noticeable difference in their biodistribution. The best variant for imaging of expression of CAIX in disseminated cancer was (99m)Tc-(HE)3-ZCAIX:2 providing tumor uptake of 16.3 ± 0.9% ID/g and tumor-to-blood ratio of 44 ± 7 at 4 h after injection. For primary renal cell carcinoma, the most promising imaging candidate was (125)I-ZCAIX:4 providing tumor-kidney ratio of 2.1 ± 0.5. In conclusion, several clones of scaffold proteins should be evaluated to select the best variant for development of an imaging probe with optimal sensitivity for the intended application.

  3. Gallium-68-labeled affibody molecule for PET imaging of PDGFRβ expression in vivo.

    PubMed

    Strand, Joanna; Varasteh, Zohreh; Eriksson, Olof; Abrahmsen, Lars; Orlova, Anna; Tolmachev, Vladimir

    2014-11-03

    Platelet-derived growth factor receptor β (PDGFRβ) is a transmembrane tyrosine kinase receptor involved, for example, in angiogenesis. Overexpression and excessive signaling of PDGFRβ has been observed in multiple malignant tumors and fibrotic diseases, making this receptor a pharmaceutical target for monoclonal antibodies and tyrosine kinase inhibitors. Successful targeted therapy requires identification of responding patients. Radionuclide molecular imaging would enable determination of the PDGFRβ status in all lesions using a single noninvasive repeatable procedure. Recently, we have demonstrated that the affibody molecule Z09591 labeled with (111)In can specifically target PDGFRβ-expressing tumors in vivo. The use of positron emission tomography (PET) as an imaging technique would provide superior resolution, sensitivity, and quantitation accuracy. In this study, a DOTA-conjugated Z09591 was labeled with the generator-produced positron emitting radionuclide (68)Ga (T1/2 = 67.6 min, Eβ + max = 1899 keV, 89% β(+)). (68)Ga-DOTA-Z09591 retained the capacity to specifically bind to PDGFRβ-expressing U-87 MG glioma cells. The half-maximum inhibition concentration (IC50) of (68)Ga-DOTA-Z09591 (6.6 ± 1.4 nM) was somewhat higher than that of (111)In-DOTA-Z09591 (1.4 ± 1.2 nM). (68)Ga-DOTA-Z09591 demonstrated specific (saturable) targeting of U-87 MG xenografts in immunodeficient mice. The tumor uptake at 2 h after injection was 3.7 ± 1.7% IA/g, which provided a tumor-to-blood ratio of 8.0 ± 3.1. The only organ with higher accumulation of radioactivity was the kidney. MicroPET imaging provided high-contrast imaging of U-87 MG xenografts. In conclusion, the (68)Ga-labeled affibody molecule Z09591 is a promising candidate for further development as a probe for imaging PDGFRβ expression in vivo using PET.

  4. In vivo evaluation of a novel format of a bivalent HER3-targeting and albumin-binding therapeutic affibody construct

    PubMed Central

    Bass, Tarek Z.; Rosestedt, Maria; Mitran, Bogdan; Frejd, Fredrik Y.; Löfblom, John; Tolmachev, Vladimir; Ståhl, Stefan; Orlova, Anna

    2017-01-01

    Overexpression of human epidermal growth factor receptor 3 (HER3) is involved in resistance to several therapies for malignant tumours. Currently, several anti-HER3 monoclonal antibodies are under clinical development. We introduce an alternative approach to HER3-targeted therapy based on engineered scaffold proteins, i.e. affibody molecules. We designed a small construct (22.5 kDa, denoted 3A3), consisting of two high-affinity anti-HER3 affibody molecules flanking an albumin-binding domain ABD, which was introduced for prolonged residence in circulation. In vitro, 3A3 efficiently inhibited growth of HER3-expressing BxPC-3 cells. Biodistribution in mice was measured using 3A3 that was site-specifically labelled with 111In via a DOTA chelator. The residence time of 111In-DOTA-3A3 in blood was extended when compared with the monomeric affibody molecule. 111In-DOTA-3A3 accumulated specifically in HER3-expressing BxPC-3 xenografts in mice. However, 111In-DOTA-3A3 cleared more rapidly from blood than a size-matched control construct 111In-DOTA-TAT, most likely due to sequestering of 3A3 by mErbB3, the murine counterpart of HER3. Repeated dosing and increase of injected protein dose decreased uptake of 111In-DOTA-3A3 in mErbB3-expressing tissues. Encouragingly, growth of BxPC-3 xenografts in mice was delayed in an experimental (pilot-scale) therapy study using 3A3. We conclude that the 3A3 affibody format seems promising for treatment of HER3-overexpressing tumours. PMID:28230065

  5. Affibody molecules: new protein domains for molecular imaging and targeted tumor therapy.

    PubMed

    Nilsson, Fredrik Y; Tolmachev, Vladimir

    2007-03-01

    Molecular imaging shows promise as a useful tool to aid drug discovery and development and also to provide important prognostic and predictive diagnostic information affecting patient management in the clinic. However, the use of molecular imaging diagnostically is not widely adopted, in part due to the lack of suitable targeting agents. Affibody molecules are a class of small and very stable protein domains, which can be used to selectively address a wide range of protein targets. Their small size enables high contrast radionuclide imaging and they can be produced by conventional peptide synthesis methods. Their potential utility in molecular imaging is highlighted in a large number of animal studies using anti-HER2 Affibody tracers and has recently been validated in breast cancer patients with HER2-expressing metastases. The therapeutic efficacy of the Affibody molecules in this indication was demonstrated in preclinical models using a targeted radionuclide as the effector function. This review will focus on the recent use of Affibody molecules for molecular imaging and their application for radioimmunotherapy.

  6. Imaging of insulinlike growth factor type 1 receptor in prostate cancer xenografts using the affibody molecule 111In-DOTA-ZIGF1R:4551.

    PubMed

    Tolmachev, Vladimir; Malmberg, Jennie; Hofström, Camilla; Abrahmsén, Lars; Bergman, Thomas; Sjöberg, Anna; Sandström, Mattias; Gräslund, Torbjörn; Orlova, Anna

    2012-01-01

    One of the pathways leading to androgen independence in prostate cancer involves upregulation of insulinlike growth factor type 1 receptor (IGF-1R). Radionuclide imaging of IGF-1R in tumors might be used for selection of patients who would most likely benefit from IGF-1R-targeted therapy. The goal of this study was to evaluate the feasibility of in vivo radionuclide imaging of IGF-1R expression in prostate cancer xenografts using a small nonimmunoglobulin-derived binding protein called an Affibody molecule. The IGF-1R-binding Z(IGF1R:4551) Affibody molecule was site-specifically conjugated with a maleimido derivative of DOTA and labeled with (111)In. The binding of radiolabeled Z(IGF1R:4551) to IGF-1R-expressing cells was evaluated in vitro and in vivo. DOTA-Z(IGF1R:4551) can be stably labeled with (111)In with preserved specific binding to IGF-1R-expressing cells in vitro. In mice, (111)In-DOTA-Z(IGF1R:4551) accumulated in IGF-1R-expressing organs (pancreas, stomach, lung, and salivary gland). Receptor saturation experiments demonstrated that targeting of DU-145 prostate cancer xenografts in NMRI nu/nu mice was IGF-1R-specific. The tumor uptake was 1.1 ± 0.3 percentage injected dose per gram, and the tumor-to-blood ratio was 3.2 ± 0.2 at 8 h after injection. This study demonstrates the feasibility of in vivo targeting of IGF-1R-expressing prostate cancer xenografts using an Affibody molecule. Further development of radiolabeled Affibody molecules might provide a useful clinical tool for stratification of patients with prostate cancer for IGF-1R-targeting therapy.

  7. Selection of an optimal cysteine-containing peptide-based chelator for labeling of affibody molecules with (188)Re.

    PubMed

    Altai, Mohamed; Honarvar, Hadis; Wållberg, Helena; Strand, Joanna; Varasteh, Zohreh; Rosestedt, Maria; Orlova, Anna; Dunås, Finn; Sandström, Mattias; Löfblom, John; Tolmachev, Vladimir; Ståhl, Stefan

    2014-11-24

    Affibody molecules constitute a class of small (7 kDa) scaffold proteins that can be engineered to have excellent tumor targeting properties. High reabsorption in kidneys complicates development of affibody molecules for radionuclide therapy. In this study, we evaluated the influence of the composition of cysteine-containing C-terminal peptide-based chelators on the biodistribution and renal retention of (188)Re-labeled anti-HER2 affibody molecules. Biodistribution of affibody molecules containing GGXC or GXGC peptide chelators (where X is G, S, E or K) was compared with biodistribution of a parental affibody molecule ZHER2:2395 having a KVDC peptide chelator. All constructs retained low picomolar affinity to HER2-expressing cells after labeling. The biodistribution of all (188)Re-labeled affibody molecules was in general comparable, with the main observed difference found in the uptake and retention of radioactivity in excretory organs. The (188)Re-ZHER2:V2 affibody molecule with a GGGC chelator provided the lowest uptake in all organs and tissues. The renal retention of (188)Re-ZHER2:V2 (3.1 ± 0.5 %ID/g at 4 h after injection) was 55-fold lower than retention of the parental (188)Re-ZHER2:2395 (172 ± 32 %ID/g). We show that engineering of cysteine-containing peptide-based chelators can be used for significant improvement of biodistribution of (188)Re-labeled scaffold proteins, particularly reduction of their uptake in excretory organs. Copyright © 2014 The Authors. Published by Elsevier Masson SAS.. All rights reserved.

  8. Evaluation of the radiocobalt-labeled [MMA-DOTA-Cys61]-Z HER2:2395(-Cys) affibody molecule for targeting of HER2-expressing tumors.

    PubMed

    Wållberg, Helena; Ahlgren, Sara; Widström, Charles; Orlova, Anna

    2010-01-01

    Imaging using positron emission tomography (PET) in the field of nuclear medicine is becoming increasingly important. The aim of this study was to develop a method for labeling of affibody molecules with radiocobalt for PET applications. The human epidermal growth factor receptors type 2 (HER2) binding affibody molecule DOTA-Z(2395)-C was radiolabeled with (57)Co (used as a surrogate of (55)Co). The binding specificity and cellular processing of the labeled compound was studied in vitro followed by in vivo characterization in normal and tumor-bearing mice. Furthermore, a comparative biodistribution study was performed with a (111)In-labeled counterpart. DOTA-Z(2395)-C was successfully labeled with radiocobalt with nearly quantitative yield. The compound displayed good retention on cells over time and high tumor accumulation of radioactivity in animal studies. Imaging studies showed clear visualization of HER2-positive tumors. Furthermore, the radiocobalt label provided better tumor-to-organ ratios than (111)In. Radiocobalt is a promising label for affibody molecules for future PET applications.

  9. Site-Specific Radioiodination of HER2-Targeting Affibody Molecules using 4-Iodophenethylmaleimide Decreases Renal Uptake of Radioactivity

    PubMed Central

    Strand, Joanna; Nordeman, Patrik; Honarvar, Hadis; Altai, Mohamed; Orlova, Anna; Larhed, Mats; Tolmachev, Vladimir

    2015-01-01

    Affibody molecules are small scaffold-based affinity proteins with promising properties as probes for radionuclide-based molecular imaging. However, a high reabsorption of radiolabeled Affibody molecules in kidneys is an issue. We have shown that the use of 125I-3-iodo-((4-hydroxyphenyl)ethyl)maleimide (IHPEM) for site-specific labeling of cysteine-containing Affibody molecules provides high tumor uptake but low radioactivity retention in kidneys. We hypothesized that the use of 4-iodophenethylmaleimide (IPEM) would further reduce renal retention of radioactivity because of higher lipophilicity of radiometabolites. An anti-human epidermal growth factor receptor type 2 (HER2) Affibody molecule (ZHER2:2395) was labeled using 125I-IPEM with an overall yield of 45±3 %. 125I-IPEM-ZHER2:2395 bound specifically to HER2-expressing human ovarian carcinoma cells (SKOV-3 cell line). In NMRI mice, the renal uptake of 125I-IPEM-ZHER2:2395 (24±2 and 5.7±0.3 % IA g−1at 1 and 4 h after injection, respectively) was significantly lower than uptake of 125I-IHPEM-ZHER2:2395 (50±8 and 12±2 % IA g−1at 1 and 4 h after injection, respectively). In conclusion, the use of a more lipophilic linker for the radioiodination of Affibody molecules reduces renal radioactivity. PMID:25969816

  10. Site-Specific Radioiodination of HER2-Targeting Affibody Molecules using 4-Iodophenethylmaleimide Decreases Renal Uptake of Radioactivity.

    PubMed

    Strand, Joanna; Nordeman, Patrik; Honarvar, Hadis; Altai, Mohamed; Orlova, Anna; Larhed, Mats; Tolmachev, Vladimir

    2015-04-01

    Affibody molecules are small scaffold-based affinity proteins with promising properties as probes for radionuclide-based molecular imaging. However, a high reabsorption of radiolabeled Affibody molecules in kidneys is an issue. We have shown that the use of (125)I-3-iodo-((4-hydroxyphenyl)ethyl)maleimide (IHPEM) for site-specific labeling of cysteine-containing Affibody molecules provides high tumor uptake but low radioactivity retention in kidneys. We hypothesized that the use of 4-iodophenethylmaleimide (IPEM) would further reduce renal retention of radioactivity because of higher lipophilicity of radiometabolites. An anti-human epidermal growth factor receptor type 2 (HER2) Affibody molecule (ZHER2:2395) was labeled using (125)I-IPEM with an overall yield of 45±3 %. (125)I-IPEM-ZHER2:2395 bound specifically to HER2-expressing human ovarian carcinoma cells (SKOV-3 cell line). In NMRI mice, the renal uptake of (125)I-IPEM-ZHER2:2395 (24±2 and 5.7±0.3 % IA g(-1)at 1 and 4 h after injection, respectively) was significantly lower than uptake of (125)I-IHPEM-ZHER2:2395 (50±8 and 12±2 % IA g(-1)at 1 and 4 h after injection, respectively). In conclusion, the use of a more lipophilic linker for the radioiodination of Affibody molecules reduces renal radioactivity.

  11. Cy5.5-labeled Affibody molecule for near-infrared fluorescent optical imaging of epidermal growth factor receptor positive tumors

    NASA Astrophysics Data System (ADS)

    Miao, Zheng; Ren, Gang; Liu, Hongguang; Jiang, Lei; Cheng, Zhen

    2010-05-01

    Affibody protein is an engineered protein scaffold with a three-helical bundle structure. Affibody molecules of small size (7 kD) have great potential for targeting overexpressed cancer biomarkers in vivo. To develop an Affibody-based molecular probe for in vivo optical imaging of epidermal growth factor receptor (EGFR) positive tumors, an anti-EGFR Affibody molecule, Ac-Cys-ZEGFR:1907 (7 kD), is site-specifically conjugated with a near-IR fluorescence dye, Cy5.5-mono-maleimide. Using fluorescent microscopy, the binding specificity of the probe Cy5.5-ZEGFR:1907 is checked by a high-EGFR-expressing A431 cell and low-EGFR-expressing MCF7 cells. The binding affinity of Cy5.5-ZEGFR:1907 (KD) to EGFR is 43.6+/-8.4 nM, as determined by flow cytometry. For an in vivo imaging study, the probe shows fast tumor targeting and good tumor contrast as early as 0.5 h postinjection (p.i.) for A431 tumors, while MCF7 tumors are barely visible. An ex vivo imaging study also demonstrates that Cy5.5-ZEGFR:1907 has high tumor, liver, and kidney uptakes at 24 h p.i.. In conclusion, Cy5.5-ZEGFR:1907 shows good affinity and high specificity to the EGFR. There is rapid achievement of good tumor-to-normal-tissue contrasts of Cy5.5-ZEGFR:1907, thus demonstrating its potential for EGFR-targeted molecular imaging of cancers.

  12. Influence of macrocyclic chelators on the targeting properties of (68)Ga-labeled synthetic affibody molecules: comparison with (111)In-labeled counterparts.

    PubMed

    Strand, Joanna; Honarvar, Hadis; Perols, Anna; Orlova, Anna; Selvaraju, Ram Kumar; Karlström, Amelie Eriksson; Tolmachev, Vladimir

    2013-01-01

    Affibody molecules are a class of small (7 kDa) non-immunoglobulin scaffold-based affinity proteins, which have demonstrated substantial potential as probes for radionuclide molecular imaging. The use of positron emission tomography (PET) would further increase the resolution and quantification accuracy of Affibody-based imaging. The rapid in vivo kinetics of Affibody molecules permit the use of the generator-produced radionuclide (68)Ga (T1/2=67.6 min). Earlier studies have demonstrated that the chemical nature of chelators has a substantial influence on the biodistribution properties of Affibody molecules. To determine an optimal labeling approach, the macrocyclic chelators 1,4,7,10-tetraazacylododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-N,N,N-triacetic acid (NOTA) and 1-(1,3-carboxypropyl)-1,4,7- triazacyclononane-4,7-diacetic acid (NODAGA) were conjugated to the N-terminus of the synthetic Affibody molecule ZHER2:S1 targeting HER2. Affibody molecules were labeled with (68)Ga, and their binding specificity and cellular processing were evaluated. The biodistribution of (68)Ga-DOTA-ZHER2:S1, (68)Ga-NOTA-ZHER2:S1 and (68)Ga-NODAGA-ZHER2:S1, as well as that of their (111)In-labeled counterparts, was evaluated in BALB/C nu/nu mice bearing HER2-expressing SKOV3 xenografts. The tumor uptake for (68)Ga-DOTA-ZHER2:S1 (17.9 ± 0.7%IA/g) was significantly higher than for both (68)Ga-NODAGA-ZHER2:S1 (16.13 ± 0.67%IA/g) and (68)Ga-NOTA-ZHER2:S1 (13 ± 3%IA/g) at 2 h after injection. (68)Ga-NODAGA-ZHER2:S1 had the highest tumor-to-blood ratio (60 ± 10) in comparison with both (68)Ga-DOTA-ZHER2:S1 (28 ± 4) and (68)Ga-NOTA-ZHER2:S1 (42 ± 11). The tumor-to-liver ratio was also higher for (68)Ga-NODAGA-ZHER2:S1 (7 ± 2) than the DOTA and NOTA conjugates (5.5 ± 0.6 vs.3.3 ± 0.6). The influence of chelator on the biodistribution and targeting properties was less pronounced for (68)Ga than for (111)In. The results of this study demonstrate that

  13. PET imaging of epidermal growth factor receptor expression in tumours using 89Zr-labelled ZEGFR:2377 affibody molecules.

    PubMed

    Garousi, Javad; Andersson, Ken G; Mitran, Bogdan; Pichl, Marie-Louise; Ståhl, Stefan; Orlova, Anna; Löfblom, John; Tolmachev, Vladimir

    2016-04-01

    Epidermal growth factor receptor (EGFR) is a transmembrane tyrosine kinase receptor, which is overexpressed in many types of cancer. The use of EGFR-targeting monoclonal antibodies and tyrosine-kinase inhibitors improves significantly survival of patients with colorectal, non-small cell lung cancer and head and neck squamous cell carcinoma. Detection of EGFR overexpression provides important prognostic and predictive information influencing management of the patients. The use of radionuclide molecular imaging would enable non-invasive repeatable determination of EGFR expression in disseminated cancer. Moreover, positron emission tomography (PET) would provide superior sensitivity and quantitation accuracy in EGFR expression imaging. Affibody molecules are a new type of imaging probes, providing high contrast in molecular imaging. In the present study, an EGFR-binding affibody molecule (ZEGFR:2377) was site-specifically conjugated with a deferoxamine (DFO) chelator and labelled under mild conditions (room temperature and neutral pH) with a positron-emitting radionuclide (89)Zr. The (89)Zr-DFO-ZEGFR:2377 tracer demonstrated specific high affinity (160 ± 60 pM) binding to EGFR-expressing A431 epidermoid carcinoma cell line. In mice bearing A431 xenografts, (89)Zr-DFO-ZEGFR:2377 demonstrated specific uptake in tumours and EGFR-expressing tissues. The tracer provided tumour uptake of 2.6 ± 0.5% ID/g and tumour-to-blood ratio of 3.7 ± 0.6 at 24 h after injection. (89)Zr-DFO-ZEGFR:2377 provides higher tumour-to-organ ratios than anti-EGFR antibody (89)Zr-DFO-cetuximab at 48 h after injection. EGFR‑expressing tumours were clearly visualized by microPET using (89)Zr-DFO-ZEGFR:2377 at both 3 and 24 h after injection. In conclusion, 8(9)Zr-DFO-ZEGFR:2377 is a potential probe for PET imaging of EGFR-expression in vivo.

  14. HER2-positive tumors imaged within 1 hour using a site-specifically 11C-labeled Sel-tagged affibody molecule.

    PubMed

    Wållberg, Helena; Grafström, Jonas; Cheng, Qing; Lu, Li; Martinsson Ahlzén, Hanna-Stina; Samén, Erik; Thorell, Jan-Olov; Johansson, Katarina; Dunås, Finn; Olofsson, Maria Hägg; Stone-Elander, Sharon; Arnér, Elias S J; Ståhl, Stefan

    2012-09-01

    A rapid, reliable method for distinguishing tumors or metastases that overexpress human epidermal growth factor receptor 2 (HER2) from those that do not is highly desired for individualizing therapy and predicting prognoses. In vivo imaging methods are available but not yet in clinical practice; new methodologies improving speed, sensitivity, and specificity are required. A HER2-binding Affibody molecule, Z(HER2:342), was recombinantly fused with a C-terminal selenocysteine-containing tetrapeptide Sel-tag, allowing site-specific labeling with either (11)C or (68)Ga, followed by biodistribution studies with small-animal PET. Dosimetry data for the 2 radiotracers were compared. Imaging of HER2-expressing human tumor xenografts was performed using the (11)C-labeled Affibody molecule. Both the (11)C- and (68)Ga-labeled tracers initially cleared rapidly from the blood, followed by a slower decrease to 4-5 percentage injected dose per gram of tissue at 1 h. Final retention in the kidneys was much lower (>5-fold) for the (11)C-labeled protein, and its overall absorbed dose was considerably lower. (11)C-Z(HER2:342) showed excellent tumor-targeting capability, with almost 10 percentage injected dose per gram of tissue in HER2-expressing tumors within 1 h. Specificity was demonstrated by preblocking binding sites with excess ligand, yielding significantly reduced radiotracer uptake (P = 0.002), comparable to uptake in tumors with low HER2 expression. To our knowledge, the Sel-tagging technique is the first that enables site-specific (11)C-radiolabeling of proteins. Here we present the finding that, in a favorable combination between radionuclide half-life and in vivo pharmacokinetics of the Affibody molecules, (11)C-labeled Sel-tagged Z(HER2:342) can successfully be used for rapid and repeated PET studies of HER2 expression in tumors.

  15. The influence of Bz-DOTA and CHX-A''-DTPA on the biodistribution of ABD-fused anti-HER2 Affibody molecules: implications for (114m)In-mediated targeting therapy.

    PubMed

    Tolmachev, Vladimir; Wållberg, Helena; Andersson, Karl; Wennborg, Anders; Lundqvist, Hans; Orlova, Anna

    2009-09-01

    Affibody molecules represent a novel class of high-affinity agents for radionuclide tumour targeting. Fusion of the Affibody molecules with an albumin-binding domain (ABD) enables modification of the blood kinetics of the Affibody molecules and reduction of the renal dose. (177)Lu-CHX-A''-DTPA-ABD-(Z(HER2:342))(2), an anti-HER2 Affibody molecule-ABD fusion protein has earlier demonstrated promising results in treatment of HER2-expressing micro-xenografts in mice. The use of the in vivo generator (114m)In/(114)In as a label for ABD-fused Affibody molecules would create preconditions for efficient treatment of both micrometastases (due to conversion and Auger electrons of (114m)In) and bulky tumours (due to high-energy beta particles from the daughter nuclide (114)In). The goal of this study was to investigate if different chelators influence the biodistribution of ABD-(Z(HER2:342))(2) and to find an optimal chelator for attachment of (114m)In to the Affibody molecule-ABD fusion protein. Isothiocyanate derivatives of Bz-DOTA and CHX-A''-DTPA were coupled to ABD-(Z(HER2:342))(2). The cellular processing of both conjugates was studied in vitro. The influence of chelators on the biodistribution was investigated in mice using double isotope ((114m)In and (111)In) labelling. The apparent affinity of CHX-A''-DTPA-ABD-(Z(HER2:342))(2) and Bz-DOTA-ABD-(Z(HER2:342))(2) to the extracellular domain of HER2 was similar, 13.5 and 15.0 pM, respectively. It was found that both conjugates were internalized by SKOV-3 cells. The use of CHX-A''-DTPA provided better cellular retention of the radioactivity, better tumour accumulation of radioactivity and better tumour to organ dose ratios than Bz-DOTA-ABD-(Z(HER2:342))(2). CHX-A''-DTPA is more suitable for (114m)In labelling of Affibody molecule-ABD fusion proteins for radionuclide therapy.

  16. Preclinical evaluation of anti-HER2 Affibody molecules site-specifically labeled with 111In using a maleimido derivative of NODAGA.

    PubMed

    Altai, Mohamed; Perols, Anna; Karlström, Amelie Eriksson; Sandström, Mattias; Boschetti, Frederic; Orlova, Anna; Tolmachev, Vladimir

    2012-05-01

    Affibody molecules have demonstrated potential for radionuclide molecular imaging. The aim of this study was to synthesize and evaluate a maleimido derivative of the 1,4,7-triazacyclononane-1-glutaric acid-4,7-diacetic acid (NODAGA) for site-specific labeling of anti-HER2 Affibody molecule. The maleimidoethylmonoamide NODAGA (MMA-NODAGA) was synthesized and conjugated to Z(HER2:2395) Affibody molecule having a C-terminal cysteine. Labeling efficiency, binding specificity to and cell internalization by HER2-expressing cells of [(111)In-MMA-NODAGA-Cys(61)]-Z(HER2:2395) were studied. Biodistribution of [(111)In-MMA-NODAGA-Cys(61)]-Z(HER2:2395) and [(111)In-MMA-DOTA-Cys(61)]-Z(HER2:2395) was compared in mice. The affinity of [MMA-NODAGA-Cys(61)]-Z(HER2:2395) binding to HER2 was 67 pM. The (111)In-labeling yield was 99.6%±0.5% after 30 min at 60°C. [(111)In-MMA-NODAGA-Cys(61)]-Z(HER2:2395) bound specifically to HER2-expressing cells in vitro and in vivo. Tumor uptake of [(111)In-MMA-NODAGA-Cys(61)]-Z(HER2:2395) in mice bearing DU-145 xenografts (4.7%±0.8% ID/g) was lower than uptake of [(111)In-MMA-DOTA-Cys(61)]-Z(HER2:2395) (7.5%±1.6% ID/g). However, tumor-to-organ ratios were higher for [(111)In-MMA-NODAGA-Cys(61)]-Z(HER2:2395) due to higher clearance rate from normal tissues. MMA-NODAGA is a promising chelator for site-specific labeling of targeting proteins containing unpaired cysteine. Appreciable influence of chelators on targeting properties of Affibody molecules was demonstrated. Copyright © 2012 Elsevier Inc. All rights reserved.

  17. An in vitro selected binding protein (affibody) shows conformation-dependent recognition of the respiratory syncytial virus (RSV) G protein.

    PubMed

    Hansson, M; Ringdahl, J; Robert, A; Power, U; Goetsch, L; Nguyen, T N; Uhlén, M; Ståhl, S; Nygren, P A

    1999-03-01

    Using phage-display technology, a novel binding protein (Z-affibody) showing selective binding to the RSV (Long strain) G protein was selected from a combinatorial library of a small alpha-helical protein domain (Z), derived from staphylococcal protein A (SPA). Biopanning of the Z-library against a recombinant fusion protein comprising amino acids 130-230 of the G protein from RSV-subgroup A, resulted in the selection of a Z-affibody (Z(RSV1)) which showed G protein specific binding. Using biosensor technology, the affinity (K(D)) between Z(RSV1) and the recombinant protein was determined to be in the micromolar range (10(-6) M). Interestingly, the Z(RSV1) affibody was demonstrated to also recognize the partially (54%) homologous G protein of RSV subgroup B with similar affinity. Using different recombinant RSV G protein derived fragments, the binding was found to be dependent on the presence of the cysteinyl residues proposed to be involved in the formation of an intramolecular disulfide-constrained loop structure, indicating a conformation-dependent binding. Results from epitope mapping studies, employing a panel of monoclonal antibodies directed to different RSV G protein subfragments, suggest that the Z(RSV1) affibody binding site is located within the region of amino acids 164-186 of the G protein. This region contains a 13 amino acid residue sequence which is totally conserved between subgroups A and B of RSV and extends into the cystein loop region (amino acids 173-186). The potential use of the RSV G protein-specific Z(RSV1) affibody in diagnostic and therapeutic applications is discussed.

  18. Increasing the Net Negative Charge by Replacement of DOTA Chelator with DOTAGA Improves the Biodistribution of Radiolabeled Second-Generation Synthetic Affibody Molecules.

    PubMed

    Westerlund, Kristina; Honarvar, Hadis; Norrström, Emily; Strand, Joanna; Mitran, Bogdan; Orlova, Anna; Eriksson Karlström, Amelie; Tolmachev, Vladimir

    2016-05-02

    A promising strategy to enable patient stratification for targeted therapies is to monitor the target expression in a tumor by radionuclide molecular imaging. Affibody molecules (7 kDa) are nonimmunoglobulin scaffold proteins with a 25-fold smaller size than intact antibodies. They have shown an apparent potential as molecular imaging probes both in preclinical and clinical studies. Earlier, we found that hepatic uptake can be reduced by the incorporation of negatively charged purification tags at the N-terminus of Affibody molecules. We hypothesized that liver uptake might similarly be reduced by positioning the chelator at the N-terminus, where the chelator-radionuclide complex will provide negative charges. To test this hypothesis, a second generation synthetic anti-HER2 ZHER2:2891 Affibody molecule was synthesized and labeled with (111)In and (68)Ga using DOTAGA and DOTA chelators. The chelators were manually coupled to the N-terminus of ZHER2:2891 forming an amide bond. Labeling DOTAGA-ZHER2:2891 and DOTA-ZHER2:2891 with (68)Ga and (111)In resulted in stable radioconjugates. The tumor-targeting and biodistribution properties of the (111)In- and (68)Ga-labeled conjugates were compared in SKOV-3 tumor-bearing nude mice at 2 h postinjection. The HER2-specific binding of the radioconjugates was verified both in vitro and in vivo. Using the DOTAGA chelator gave significantly lower radioactivity in liver and blood for both radionuclides. The (111)In-labeled conjugates showed more rapid blood clearance than the (68)Ga-labeled conjugates. The most pronounced influence of the chelators was found when they were labeled with (68)Ga. The DOTAGA chelator gave significantly higher tumor-to-blood (61 ± 6 vs 23 ± 5, p < 0.05) and tumor-to-liver (10.4 ± 0.6 vs 4.5 ± 0.5, p < 0.05) ratios than the DOTA chelator. This study demonstrated that chelators may be used to alter the uptake of Affibody molecules, and most likely other scaffold-based imaging probes, for improvement

  19. 17AAG-induced internalisation of HER2-specific Affibody molecules

    PubMed Central

    Göstring, Lovisa; Lindegren, Sture; Gedda, Lars

    2016-01-01

    The geldanamycin derivative 17-allylamino-17-demethoxygeldanamycin (17-AAG) is known to induce internalisation and degradation of the otherwise internalisation-resistant human epidermal growth factor receptor 2 (HER2) receptor. In the present study, 17-AAG was used to increase internalisation of the HER2-specific Affibody molecule ABY-025. The cellular redistribution of halogen-labelled 211At-ABY-025 and radiometal-labelled 111In-ABY-025 following treatment with 17-AAG was studied. 17-AAG treatment of SKOV-3 human ovarian carcinoma and SKBR-3 human breast carcinoma cells to some extent shifted the localisation of 111In-ABY-025 from the cell surface to intracellular compartments in the two cell lines. ABY-025 labelled with the high-linear energy transfer α emitter 211At was also internalised to a higher degree; however, due to its physiological properties, this nuclide was excreted faster. The results indicate that 17-AAG may be used to facilitate cell-specific intracellular localisation of a suitable cytotoxic or radioactive agent coupled to ABY-025 in HER2-overexpressing cells. PMID:27698830

  20. Influence of an aliphatic linker between DOTA and synthetic Z(HER2:342) Affibody molecule on targeting properties of the (111)In-labeled conjugate.

    PubMed

    Tolmachev, Vladimir; Feldwisch, Joachim; Lindborg, Malin; Baastrup, Barbro; Sandström, Mattias; Orlova, Anna

    2011-07-01

    Affibody molecules are small (∼6.5 kDa) scaffold proteins suitable for radionuclide imaging of tumor-associated molecular targets. Site-specific labeling of Affibody molecules made by peptide synthesis can be achieved by coupling a chelator to N-terminus in the last synthesis step. The goal of this study was to evaluate the influence of a 6-aminohexanoic linker between DOTA and Z(HER2:342) on targeting properties of (111)In-labeled conjugate. A DOTA-conjugated 6-aminohexanoic linker-containing variant of Z(HER2:342) (ABY-003) was produced by peptide synthesis, and the in vitro binding affinity, specificity and cellular processing were evaluated. The biodistribution of (111)In-ABY-003 in normal mice was compared to (111)In-ABY-002 (DOTA-Z(HER2:342-pep2)) lacking the linker. Tumor-targeting properties of (111)In-ABY-003 were evaluated in mice bearing HER2-expressing xenografts. The dissociation constant of ABY-003 was in the low picomolar range, slightly higher than for ABY-002. (111)In-ABY-003 bound specifically to HER2-expressing cells in vitro. The cellular retention was efficient but slightly worse than for (111)In-ABY-002. In normal mice, the clearance of (111)In-ABY-003 from blood and other tissues was slightly but significantly faster compared to (111)In-ABY-002. Targeting of HER2-expressing xenografts by (111)In-ABY-003 was receptor-specific. Due to faster clearance, the tumor-to-blood ratio for (111)In-ABY-003 at 4 h postinjection was improved compared to (111)In-ABY-002. The capacity of (111)In-ABY-003 to visualize HER2-expressing tumors was confirmed by gamma camera imaging. A 6-aminohexanoic linker between the DOTA chelator and N-terminus of synthetic Z(HER2:342) had a measurable effect on affinity, cellular retention of radioactivity and blood clearance. The linker might be used for modulation of targeting properties of Affibody molecules. Copyright © 2011 Elsevier Inc. All rights reserved.

  1. In vivo targeting of HER2-positive tumor using 2-helix affibody molecules.

    PubMed

    Ren, Gang; Webster, Jack M; Liu, Zhe; Zhang, Rong; Miao, Zheng; Liu, Hongguang; Gambhir, Sanjiv S; Syud, Faisal A; Cheng, Zhen

    2012-07-01

    Molecular imaging of human epidermal growth factor receptor type 2 (HER2) expression has drawn significant attention because of the unique role of the HER2 gene in diagnosis, therapy and prognosis of human breast cancer. In our previous research, a novel cyclic 2-helix small protein, MUT-DS, was discovered as an anti-HER2 Affibody analog with high affinity through rational protein design and engineering. MUT-DS was then evaluated for positron emission tomography (PET) of HER2-positive tumor by labeling with two radionuclides, 68Ga and 18F, with relatively short half-life (t1/2<2 h). In order to fully study the in vivo behavior of 2-helix small protein and demonstrate that it could be a robust platform for labeling with a variety of radionuclides for different applications, in this study, MUT-DS was further radiolabeled with 64Cu or 111In and evaluated for in vivo targeting of HER2-positive tumor in mice. Design 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) conjugated MUT-DS (DOTA-MUT-DS) was chemically synthesized using solid phase peptide synthesizer and I2 oxidation. DOTA-MUT-DS was then radiolabeled with 64Cu or 111In to prepare the HER2 imaging probe (64Cu/111In-DOTA-MUT-DS). Both biodistribution and microPET imaging of the probe were evaluated in nude mice bearing subcutaneous HER2-positive SKOV3 tumors. DOTA-MUT-DS could be successfully synthesized and radiolabeled with 64Cu or 111In. Biodistribution study showed that tumor uptake value of 64Cu or 111In-labeled DOTA-MUT-DS was 4.66±0.38 or 2.17±0.15%ID/g, respectively, in nude mice bearing SKOV3 xenografts (n=3) at 1 h post-injection (p.i.). Tumor-to-blood and tumor-to-muscle ratios for 64Cu-DOTA-MUT-DS were attained to be 3.05 and 3.48 at 1 h p.i., respectively, while for 111In-DOTA-MUT-DS, they were 2.04 and 3.19, respectively. Co-injection of the cold Affibody molecule ZHER2:342 with 64Cu-DOTA-MUT-DS specifically reduced the SKOV3 tumor uptake of the probe by 48%. 111In

  2. Affibody molecules for molecular imaging and targeted drug delivery in the management of breast cancer.

    PubMed

    De, Anindita; Kuppusamy, Gowthamarajan; Karri, Veera Venkata Satyanarayana Reddy

    2017-09-19

    Breast cancer is one of the leading reasons for the morbidity and mortality of cancer related death globally. The modern therapies are basically the combination of the breast-preserving surgeries or ablation with or without node biopsy or destroying the carcinoma cells adjuvant with chemotherapy, radiotherapy, hormonal or biological therapies depending upon the nature of the receptor of the cancerous cells, nature of the lymph node, as well as the tendency of the recurrence. For decade's carcinoma management suffered by the limitation of imagining, targeting and penetrability problem associated with management and cure of this deadly disease leads to unwanted chemo-toxicity and side effects. Alike other antibody mimetics, affibodies are designed with the combinatorial protein engineering approaches which are small and robust protein scaffolds retaining the favorable folding and stability. Affibody is one of the significantly important tools for imaging and diagnosis of the affinity specific over expressed proteins in the breast cancer management. The review summarizes the various affibody strategies uses in the management of breast cancer. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. Order of amino acids in C-terminal cysteine-containing peptide-based chelators influences cellular processing and biodistribution of 99mTc-labeled recombinant Affibody molecules.

    PubMed

    Altai, Mohamed; Wållberg, Helena; Orlova, Anna; Rosestedt, Maria; Hosseinimehr, Seyed Jalal; Tolmachev, Vladimir; Ståhl, Stefan

    2012-05-01

    Affibody molecules constitute a novel class of molecular display selected affinity proteins based on non-immunoglobulin scaffold. Preclinical investigations and pilot clinical data have demonstrated that Affibody molecules provide high contrast imaging of tumor-associated molecular targets shortly after injection. The use of cysteine-containing peptide-based chelators at the C-terminus of recombinant Affibody molecules enabled site-specific labeling with the radionuclide 99mTc. Earlier studies have demonstrated that position, composition and the order of amino acids in peptide-based chelators influence labeling stability, cellular processing and biodistribution of Affibody molecules. To investigate the influence of the amino acid order, a series of anti-HER2 Affibody molecules, containing GSGC, GEGC and GKGC chelators have been prepared and characterized. The affinity to HER2, cellular processing of 99mTc-labeled Affibody molecules and their biodistribution were investigated. These properties were compared with that of the previously studied 99mTc-labeled Affibody molecules containing GGSC, GGEC and GGKC chelators. All variants displayed picomolar affinities to HER2. The substitution of a single amino acid in the chelator had an appreciable influence on the cellular processing of 99mTc. The biodistribution of all 99mTc-labeled Affibody molecules was in general comparable, with the main difference in uptake and retention of radioactivity in excretory organs. The hepatic accumulation of radioactivity was higher for the lysine-containing chelators and the renal retention of 99mTc was significantly affected by the amino acid composition of chelators. The order of amino acids influenced renal uptake of some conjugates at 1 h after injection, but the difference decreased at later time points. Such information can be helpful for the development of other scaffold protein-based imaging and therapeutic radiolabeled conjugates.

  4. Imaging agents for in vivo molecular profiling of disseminated prostate cancer--targeting EGFR receptors in prostate cancer: comparison of cellular processing of [111In]-labeled affibody molecule Z(EGFR:2377) and cetuximab.

    PubMed

    Malmberg, Jennie; Tolmachev, Vladimir; Orlova, Anna

    2011-04-01

    Expression of receptor tyrosine-kinase (RTK) EGFR is low in normal prostate, but increases in prostate cancer. This receptor is significantly up-regulated as tumors progress into higher grade, androgen-insensitive and metastatic lesions. The up-regulated receptors could serve as targets for novel selective anti-cancer drugs, e.g. antibodies and tyrosine kinase inhibitors. Radionuclide imaging of RTK can facilitate patient stratification and monitoring of anti-RTK therapy of prostate cancer. The goal of the study was to evaluate binding and cellar processing of radiolabeled EGFR-targeting conjugates by prostate cancer cell lines. Receptor expression of EGFR was studied in three prostate cancer cell lines: DU145 (brain metastasis of PC, hormone insensitive), PC3 (bone metastasis of PC) and LNCaP (lymph node metastasis of PC, androgen and estrogen receptor positive). Uptake and internalization of anti-EGFR mAbs (cetuximab) and affibody molecule (Z2377) labeled with indium-111 was investigated. EGFR expression on prostate cancer cell lines was clearly demonstrated. Both labelled conjugates 111In-Z2377 and 111In-cetuximab bound to prostate cancer cells in the receptor mediated model. Expression levels were modest but correlate with degree of hormone independence. Internalization of Affibody molecules was relatively slow in all cell lines. Internalization of mAbs was more rapid. The level of EGFR expression in these cell lines is sufficient for in vivo molecular imaging. Slow internalization indicates possibility of the use of non-residualizing labels for affibody molecules.

  5. Imaging of HER2-expressing tumours using a synthetic Affibody molecule containing the 99mTc-chelating mercaptoacetyl-glycyl-glycyl-glycyl (MAG3) sequence.

    PubMed

    Engfeldt, Torun; Orlova, Anna; Tran, Thuy; Bruskin, Alexander; Widström, Charles; Karlström, Amelie Eriksson; Tolmachev, Vladimir

    2007-05-01

    Expression of human epidermal growth factor receptor type 2 (HER2) in malignant tumours possesses well-documented prognostic and predictive value. Non-invasive imaging of expression can provide valuable diagnostic information, thereby influencing patient management. Previously, we reported a phage display selection of a small (about 7 kDa) protein, the Affibody molecule Z(HER2:342), which binds HER2 with subnanomolar affinity, and demonstrated the feasibility of targeting of HER2-expressing xenografts using radioiodinated Z(HER2:342). The goal of this study was to develop a method for (99m)Tc labelling of Z(HER2:342) using the MAG3 chelator, which was incorporated into Z(HER2:342) using peptide synthesis, and evaluate the targeting properties of the labelled conjugate. MAG3-Z(HER2:342) was assembled using Fmoc/tBu solid phase peptide synthesis. Biochemical characterisation of the agent was performed using RP-HPLC, ESI-MS, biosensor studies and circular dichroism. A procedure for (99m)Tc labelling in the presence of sodium/potassium tartrate was established. Tumour targeting was evaluated by biodistribution study and gamma camera imaging in xenograft-bearing mice. Biodistribution of (99m)Tc-MAG3-Z(HER2:342) and (125)I-para-iodobenzoate -Z(HER2:342) was compared 6 h p.i. Synthetic MAG3-Z(HER2:342) possessed an affinity of 0.2 nM for HER2 receptors. The peptide was labelled with (99m)Tc with an efficiency of about 75-80%. Labelled (99m)Tc-MAG3-Z(HER2:342) retained capacity to bind specifically HER2-expressing SKOV-3 cells in vitro. (99m)Tc-MAG3-Z(HER2:342) showed specific tumour targeting with a contrast similar to a radioiodinated analogue in mice bearing LS174T xenografts. Gamma camera imaging demonstrated clear and specific visualisation of HER2 expression. Incorporation of a mercaptoacetyl-containing chelating sequence during chemical synthesis enabled site-specific (99m)Tc labelling of the Z(HER2:342) Affibody molecule with preserved targeting capacity.

  6. Target-specific cytotoxic effects on HER2-expressing cells by the tripartite fusion toxin ZHER2:2891-ABD-PE38X8, including a targeting affibody molecule and a half-life extension domain.

    PubMed

    Liu, Hao; Seijsing, Johan; Frejd, Fredrik Y; Tolmachev, Vladimir; Gräslund, Torbjörn

    2015-08-01

    Development of cancer treatment regimens including immunotoxins is partly hampered by their immunogenicity. Recently, deimmunized versions of toxins have been described, potentially being better suited for translation to the clinic. In this study, a recombinant tripartite fusion toxin consisting of a deimmunized version of exotoxin A from Pseudomonas aeruginosa (PE38) genetically fused to an affibody molecule specifically interacting with the human epidermal growth factor receptor 2 (HER2), and also an albumin binding domain (ABD) for half-life extension, has been produced and characterized in terms of functionality of the three moieties. Biosensor based assays showed that the fusion toxin was able to interact with human and mouse serum albumin, but not with bovine serum albumin and that it interacted with HER2 (KD=5 nM). Interestingly, a complex of the fusion toxin and human serum albumin also interacted with HER2 but with a somewhat weaker affinity (KD=12 nM). The IC50-values of the fusion toxin ranged from 6 to 300 pM on SKOV-3, SKBR-3 and A549 cells and was lower for cells with higher surface densities of HER2. The fusion toxin was found specific for HER2 as shown by blocking available HER2 receptors with free affibody molecule before subjecting the cells to the toxin. Analysis of contact time showed that 10 min was sufficient to kill 50% of the cells. In conclusion, all three regions of the fusion toxin were found to be functional.

  7. Imaging of EGFR expression in murine xenografts using site-specifically labelled anti-EGFR 111In-DOTA-Z EGFR:2377 Affibody molecule: aspect of the injected tracer amount.

    PubMed

    Tolmachev, Vladimir; Rosik, Daniel; Wållberg, Helena; Sjöberg, Anna; Sandström, Mattias; Hansson, Monika; Wennborg, Anders; Orlova, Anna

    2010-03-01

    Overexpression of epidermal growth factor receptor (EGFR) is a prognostic and predictive biomarker in a number of malignant tumours. Radionuclide molecular imaging of EGFR expression in cancer could influence patient management. However, EGFR expression in normal tissues might complicate in vivo imaging. The aim of this study was to evaluate if optimization of the injected protein dose might improve imaging of EGFR expression in tumours using a novel EGFR-targeting protein, the DOTA-Z(EGFR:2377) Affibody molecule. An anti-EGFR Affibody molecule, Z(EGFR:2377), was labelled with (111)In via the DOTA chelator site-specifically conjugated to a C-terminal cysteine. The affinity of DOTA-Z(EGFR:2377) for murine and human EGFR was measured by surface plasmon resonance. The cellular processing of (111)In-DOTA-Z(EGFR:2377) was evaluated in vitro. The biodistribution of radiolabelled Affibody molecules injected in a broad range of injected Affibody protein doses was evaluated in mice bearing EGFR-expressing A431 xenografts. Site-specific coupling of DOTA provided a uniform conjugate possessing equal affinity for human and murine EGFR. The internalization of (111)In-DOTA-Z(EGFR:2377) by A431 cells was slow. In vivo, the conjugate accumulated specifically in xenografts and in EGFR-expressing tissues. The curve representing the dependence of tumour uptake on the injected Affibody protein dose was bell-shaped. The highest specific radioactivity (lowest injected protein dose) provided a suboptimal tumour-to-blood ratio. The results of the biodistribution study were confirmed by gamma-camera imaging. The (111)In-DOTA-Z(EGFR:2377) Affibody molecule is a promising tracer for radionuclide molecular imaging of EGFR expression in malignant tumours. Careful optimization of protein dose is required for high-contrast imaging of EGFR expression in vivo.

  8. Feasibility of imaging of epidermal growth factor receptor expression with ZEGFR:2377 affibody molecule labeled with 99mTc using a peptide-based cysteine-containing chelator

    PubMed Central

    Andersson, Ken G.; Oroujeni, Maryam; Garousi, Javad; Mitran, Bogdan; Ståhl, Stefan; Orlova, Anna; Löfblom, John; Tolmachev, Vladimir

    2016-01-01

    The epidermal growth factor receptor (EGFR) is overexpressed in a number of malignant tumors and is a molecular target for several specific anticancer antibodies and tyrosine kinase inhibitors. The overexpression of EGFR is a predictive biomarker for response to several therapy regimens. Radionuclide molecular imaging might enable detection of EGFR overexpression by a non-invasive procedure and could be used repeatedly. Affibody molecules are engineered scaffold proteins, which could be selected to have a high affinity and selectivity to predetermined targets. The anti-EGFR ZEGFR:2377 affibody molecule is a potential imaging probe for EGFR detection. The use of the generator-produced radionuclide 99mTc should facilitate clinical translation of an imaging probe due to its low price, availability and favorable dosimetry of the radionuclide. In the present study, we evaluated feasibility of ZEGFR:2377 labeling with 99mTc using a peptide-based cysteine-containing chelator expressed at the C-terminus of ZEGFR:2377. The label was stable in vitro under cysteine challenge. In addition, 99mTc-ZEGFR:2377 was capable of specific binding to EGFR-expressing cells with high affinity (274 pM). Studies in BALB/C nu/nu mice bearing A431 xenografts demonstrated that 99mTc-ZEGFR:2377 accumulates in tumors in an EGFR-specific manner. The tumor uptake values were 3.6±1 and 2.5±0.4% ID/g at 3 and 24 h after injection, respectively. The corresponding tumor-to-blood ratios were 1.8±0.4 and 8±3. The xenografts were clearly visualized at both time-points. This study demonstrated the potential of 99mTc-labeled ZEGFR:2377 for imaging of EGFR in vivo. PMID:27748899

  9. Affinity enhancement of HER2-binding Z(HER2:342) affibody via rational design approach: a molecular dynamics study.

    PubMed

    Ghaffari, Mohammad Ali; Zeinali, Majid; Barzegari Asadabadi, Ebrahim; Jamalan, Mostafa; Jahandideh, Samad

    2014-12-01

    Human epidermal growth factor receptor 2 (HER2) contributes to the development of breast cancers and malignancies. On the other hand, engineered affibody Z(HER2:342) that binds to HER2 can be successfully used for both diagnostic purposes and specific ablation of malignant HER2-positive cell lines. In the current study, electrostatics-based prediction was applied for improving Z(HER2:342) binding affinity using computational design. The affibody Z(HER2:342) alone and in complex with HER2 was energetically minimized, solvated in explicit water, and neutralized. After heating and equilibration steps, the system was studied by isothermal-isobaric (NPT) MD simulation. According to trajectories, Z(HER2:342) specifically binds to HER2 through hydrogen bonds and salt bridges. Based on the electrostatic binding contributions, two affinity-matured variants namely V1 (Tyr35Arg) and V2 (Asn6Asp and Met9Glu) were rationally designed. More investigations through MD simulation show that V1 interacts with HER2 receptor more strongly, compared to Z(HER2:342) and V2.

  10. Evaluation of 99mTc-peptide-ZHER2:342 Affibody® molecule for in vivo molecular imaging

    PubMed Central

    Zhang, J-M; Zhao, X-M; Ren, X-C; Wang, N; Han, J-Y; Jia, L-Z

    2014-01-01

    Objective: The aim of this study was to develop an improved method for labelling ZHER2:342 with Technetium-99m (99mTc) using Gly-(d) Ala-Gly-Gly as a chelator and to evaluate the feasibility of its use for visualization of HER2 expression in vivo. Methods: The Affibody® molecule ZHER2:342 was synthesized by Fmoc/tBu solid phase synthesis. The chelator, Gly-(d) Ala-Gly-Gly, was introduced by manual synthesis as the N-terminal extensions of ZHER2:342. ZHER2:342 was labelled with 99mTc. The labelling efficiency, radiochemical purity and in vitro stability of the labelled molecular probe were analysed by reversed-phase high performance liquid chromatography. Biodistribution and molecular imaging using 99mTc-peptide-ZHER2:342 were performed. Results: The molecular probe was successfully synthesized and labelled with 99mTc with the labelling efficiency of 98.10 ± 1.73% (n = 5). The radiolabelled molecular probe remained highly stable in vitro. The molecular imaging showed high uptake in HER2-expressing SKOV-3 xenografts, whereas the MDA-MB-231 xenografts with low HER2 expression were not clearly imaged at any time after the injection of 99mTc-peptide-ZHER2:342. The predominant clearance pathway for 99mTc-peptide-ZHER2:342 was through the kidneys. Conculsion: 99mTc-peptide-ZHER2:342 using Gly-(d) Ala-Gly-Gly as a chelator is a promising tracer agent with favourable biodistribution and imaging properties that may be developed as a radiopharmaceutical for the detection of HER2-positive malignant tumours. Advances in knowledge: The 99mTc-peptide-ZHER2:342 molecular probe is a promising tracer agent, and the results in this study provide a foundation for future development of protocols for earlier visual detection of cancer in the clinical setting. PMID:24273251

  11. In vitro characterization of a bivalent anti-HER-2 affibody with potential for radionuclide-based diagnostics.

    PubMed

    Steffen, Ann-Charlott; Wikman, Maria; Tolmachev, Vladimir; Adams, Gregory P; Nilsson, Fredrik Y; Ståhl, Stefan; Carlsson, Jörgen

    2005-06-01

    The 185 kDa transmembrane glycoprotein human epidermal growth factor receptor 2 (HER-2) (p185/neu, c-ErbB-2) is overexpressed in breast and ovarian cancers. Overexpression in breast cancer correlates with poor patient prognosis, and visualization of HER-2 expression might provide valuable diagnostic information influencing patient management. We have previously described the generation of a new type of affinity ligand, a 58-amino-acid affibody (Z(HER2:4)) with specific binding to HER-2. In order to benefit from avidity effects, we have created a bivalent form of the affibody ligand, (Z(HER2:4))2. The monovalent and bivalent ligands were compared in various assays. The new bivalent affibody has a molecular weight of 15.6 kDa and an apparent affinity (K(D)) against HER-2 of 3 nM. After radioiodination, using the linker molecule N-succinimidyl p-(trimethylstannyl) benzoate (SPMB), in vitro binding assays showed specific binding to HER-2 overexpressing cells. Internalization of 125I was shown after delivery with both the monovalent and the bivalent affibody. The cellular retention of 125I was longer after delivery with the bivalent affibody when compared to delivery with the monovalent affibody. With approximately the same affinity as the monoclonal antibody trastuzumab (Herceptin) but only one tenth of the size, this new bivalent molecule is a promising candidate for radionuclide-based detection of HER-2 expression in tumors. 125I was used in this study as a surrogate marker for the diagnostically relevant radioisotopes 123I for single photon emission computed tomography (SPECT)/gamma-camera imaging and 124I for positron emission tomography (PET).

  12. Evaluation of 99mTc-Z IGF1R:4551-GGGC affibody molecule, a new probe for imaging of insulin-like growth factor type 1 receptor expression.

    PubMed

    Mitran, Bogdan; Altai, Mohamed; Hofström, Camilla; Honarvar, Hadis; Sandström, Mattias; Orlova, Anna; Tolmachev, Vladimir; Gräslund, Torbjörn

    2015-02-01

    Overexpression of insulin-like growth factor-1 receptor (IGF-1R) in several cancers is associated with resistance to therapy. Radionuclide molecular imaging of IGF-1R expression in tumors may help in selecting the patients that will potentially respond to IGF-1R-targeted therapy. Affibody molecules are small (7 kDa) non-immunoglobulin-based scaffold proteins that are well-suited probes for radionuclide imaging. The aim of this study was the evaluation of an anti-IGF-1R affibody molecule labeled with technetium-99m using cysteine-containing peptide-based chelator GGGC at C-terminus. ZIGF1R:4551-GGGC was efficiently and stably labeled with technetium-99m (radiochemical yield 97 ± 3%). (99m)Tc-ZIGF1R:4551-GGGC demonstrated specific binding to IGF-1R-expressing DU-145 (prostate cancer) and MCF-7 (breast cancer) cell lines and slow internalization in vitro. The tumor-targeting properties were studied in BALB/c nu/nu mice bearing DU-145 and MCF-7 xenografts. [(99m)Tc(CO)3](+)-(HE)3-ZIGF1R:4551 was used for comparison. The biodistribution study demonstrated high tumor-to-blood ratios (6.2 ± 0.9 and 6.9 ± 1.0, for DU-145 and MCF-7, respectively, at 4 h after injection). Renal radioactivity concentration was 16-fold lower for (99m)Tc-ZIGF1R:4551-GGGC than for [(99m)Tc(CO)3](+)-(HE)3-ZIGF1R:4551 at 4 h after injection. However, the liver uptake of (99m)Tc-ZIGF1R:4551-GGGC was 1.2- to 2-fold higher in comparison with [(99m)Tc(CO)3](+)-(HE)3-ZIGF1R:4551. A possible reason for the elevated hepatic uptake of (99m)Tc-ZIGF1R:4551-GGGC is a high lipophilicity of amino acids in the binding site of ZIGF1R:4551, which is not compensated in (99m)Tc-ZIGF1R:4551-GGGC. In conclusion, (99m)Tc-ZIGF1R:4551-GGGC can visualize the IGF-1R expression in human tumor xenografts and provides low retention of radioactivity in kidneys. Further development of this imaging agent should include molecular design aimed at reducing the hepatic uptake.

  13. Comparative evaluation of synthetic anti-HER2 Affibody molecules site-specifically labelled with 111In using N-terminal DOTA, NOTA and NODAGA chelators in mice bearing prostate cancer xenografts.

    PubMed

    Malmberg, Jennie; Perols, Anna; Varasteh, Zohreh; Altai, Mohamed; Braun, Alexis; Sandström, Mattias; Garske, Ulrike; Tolmachev, Vladimir; Orlova, Anna; Karlström, Amelie Eriksson

    2012-03-01

    In disseminated prostate cancer, expression of human epidermal growth factor receptor type 2 (HER2) is one of the pathways to androgen independence. Radionuclide molecular imaging of HER2 expression in disseminated prostate cancer might identify patients for HER2-targeted therapy. Affibody molecules are small (7 kDa) targeting proteins with high potential as tracers for radionuclide imaging. The goal of this study was to develop an optimal Affibody-based tracer for visualization of HER2 expression in prostate cancer. A synthetic variant of the anti-HER2 Z(HER2:342) Affibody molecule, Z(HER2:S1), was N-terminally conjugated with the chelators DOTA, NOTA and NODAGA. The conjugated proteins were biophysically characterized by electrospray ionization mass spectroscopy (ESI-MS), circular dichroism (CD) spectroscopy and surface plasmon resonance (SPR)-based biosensor analysis. After labelling with (111)In, the biodistribution was assessed in normal mice and the two most promising conjugates were further evaluated for tumour targeting in mice bearing DU-145 prostate cancer xenografts. The HER2-binding equilibrium dissociation constants were 130, 140 and 90 pM for DOTA-Z(HER2:S1), NOTA-Z(HER2:S1) and NODAGA-Z(HER2:S1), respectively. A comparative study of (111)In-labelled DOTA-Z(HER2:S1), NOTA-Z(HER2:S1) and NODAGA-Z(HER2:S1) in normal mice demonstrated a substantial influence of the chelators on the biodistribution properties of the conjugates. (111)In-NODAGA-Z(HER2:S1) had the most rapid clearance from blood and healthy tissues. (111)In-NOTA-Z(HER2:S1) showed high hepatic uptake and was excluded from further evaluation. (111)In-DOTA-Z(HER2:S1) and (111)In-NODAGA-Z(HER2:S1) demonstrated specific uptake in DU-145 prostate cancer xenografts in nude mice. The tumour uptake of (111)In-NODAGA-Z(HER2:S1), 5.6 ± 0.4%ID/g, was significantly lower than the uptake of (111)In-DOTA-Z(HER2:S1), 7.4 ± 0.5%ID/g, presumably because of lower bioavailability due to more rapid clearance

  14. Evaluation of affibody molecule-based PNA-mediated radionuclide pretargeting: Development of an optimized conjugation protocol and (177)Lu labeling.

    PubMed

    Altai, Mohamed; Westerlund, Kristina; Velletta, Justin; Mitran, Bogdan; Honarvar, Hadis; Karlström, Amelie Eriksson

    2017-07-12

    We have previously developed a pretargeting approach for affibody-mediated cancer therapy based on PNA-PNA hybridization. In this article we have further developed this approach by optimizing the production of the primary agent, ZHER2:342-SR-HP1, and labeling the secondary agent, HP2, with the therapeutic radionuclide (177)Lu. We also studied the biodistribution profile of (177)Lu-HP2 in mice, and evaluated pretargeting with (177)Lu-HP2 in vitro and in vivo. The biodistribution profile of (177)Lu-HP2 was evaluated in NMRI mice and compared to the previously studied (111)In-HP2. Pretargeting using (177)Lu-HP2 was studied in vitro using the HER2-expressing cell lines BT-474 and SKOV-3, and in vivo in mice bearing SKOV-3 xenografts. Using an optimized production protocol for ZHER2:342-SR-HP1 the ligation time was reduced from 15h to 30min, and the yield increased from 45% to 70%. (177)Lu-labeled HP2 binds specifically in vitro to BT474 and SKOV-3 cells pre-treated with ZHER2:342-SR-HP1. (177)Lu-HP2 was shown to have a more rapid blood clearance compared to (111)In-HP2 in NMRI mice, and the measured radioactivity in blood was 0.22±0.1 and 0.68±0.07%ID/g for (177)Lu- and (111)In-HP2, respectively, at 1h p.i. In contrast, no significant difference in kidney uptake was observed (4.47±1.17 and 3.94±0.58%ID/g for (177)Lu- and (111)In-HP2, respectively, at 1h p.i.). Co-injection with either Gelofusine or lysine significantly reduced the kidney uptake for (177)Lu-HP2 (1.0±0.1 and 1.6±0.2, respectively, vs. 2.97±0.87%ID/g in controls at 4h p.i.). (177)Lu-HP2 accumulated in SKOV-3 xenografts in BALB/C nu/nu mice when administered after injection of ZHER2:342-SR-HP1. Without pre-injection of ZHER2:342-SR-HP1, the uptake of (177)Lu-HP2 was about 90-fold lower in tumor (0.23±0.08 vs. 20.7±3.5%ID/g). The tumor-to-kidney radioactivity accumulation ratio was almost 5-fold higher in the group of mice pre-injected with ZHER2:342-SR-HP1. In conclusion, (177)Lu-HP2 was shown to

  15. Anti-idiotypic protein domains selected from protein A-based affibody libraries.

    PubMed

    Eklund, Malin; Axelsson, Lars; Uhlén, Mathias; Nygren, Per-Ake

    2002-08-15

    Three pairs of small protein domains showing binding behavior in analogy with anti-idiotypic antibodies have been selected using phage display technology. From an affibody protein library constructed by combinatorial variegation of the Fc binding surface of the 58 residue staphylococcal protein A (SPA)-derived domain Z, affibody variants have been selected to the parental SPA scaffold and to two earlier identified SPA-derived affibodies. One selected affibody (Z(SPA-1)) was shown to recognize each of the five domains of wild-type SPA with dissociation constants (K(D)) in the micromolar range. The binding of the Z(SPA-1) affibody to its parental structure was shown to involve the Fc binding site of SPA, while the Fab-binding site was not involved. Similarly, affibodies showing anti-idiotypic binding characteristics were also obtained when affibodies previously selected for binding to Taq DNA polymerase and human IgA, respectively, were used as targets for selections. The potential applications for these types of affinity pairs were exemplified by one-step protein recovery using affinity chromatography employing the specific interactions between the respective protein pair members. These experiments included the purification of the Z(SPA-1) affibody from a total Escherichia coli cell lysate using protein A-Sepharose, suggesting that this protein A/antiprotein A affinity pair could provide a basis for novel affinity gene fusion systems. The use of this type of small, robust, and easily expressed anti-idiotypic affibody pair for affinity technology applications, including self-assembled protein networks, is discussed.

  16. Affibody-displaying bio-nanocapsules effective in EGFR, typical biomarker, expressed in various cancer cells.

    PubMed

    Nishimura, Yuya; Ezawa, Ryosuke; Ishii, Jun; Ogino, Chiaki; Kondo, Akihiko

    2017-01-15

    The expression of epidermal growth factor receptor (EGFR) across a wide range of tumor cells has attracted attention for use as a tumor marker in drug delivery systems. Therefore, binding molecules with the ability to target EGFR have been developed. Among them, we focused on affibodies that are binding proteins derived from staphylococcal protein A. By displaying affibody (ZEGFR) binding to EGFR on the surface of a bio-nanocapsule (BNC) derived from a hepatitis B virus (HBV), we developed an altered BNC (ZEGFR-BNC) with a high specificity to EGFR-expressing cells. We considered two different types of ZEGFR (Z955 and Z1907), and found that the Z1907 dimer-displaying BNC ([Z1907]2-BNC) could effectively bind to EGFR-expressing cells and deliver drugs to the cytosol. Since this study showed that [Z1907]2-BNC could target EGFR-expressing cells, we would use this particle as a drug delivery carrier for various cancer cells expressing EGFR. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

  17. Optical Imaging with HER2-targeted Affibody Molecules can monitor Hsp90 treatment response in a breast cancer xenograft mouse model

    PubMed Central

    van de Ven, Stephanie M.W.Y.; Elias, Sjoerd G.; Chan, Carmel T.; Miao, Zheng; Cheng, Zhen; De, Abhijit; Gambhir, Sanjiv S.

    2012-01-01

    Purpose To determine if optical imaging can be used for in vivo therapy response monitoring as an alternative to radionuclide techniques. For this we evaluated the known Her2 response to 17-DMAG treatment, a Hsp90 inhibitor. Experimental design After in vitro 17-DMAG treatment response evaluation of MCF7 parental cells and two HER2 transfected clones (Clone A medium, B high Her2 expression), we established human breast cancer xenografts in nude mice (only parental and clone B) for in vivo evaluation. Mice received 120 mg/kg of 17-DMAG in 4 doses at 12 hour intervals i.p. (n=14), or PBS as carrier control (n=9). Optical images were obtained both pre-treatment (day 0) and post-treatment (day 3, 6, and 9), always 5 hours post-injection of 500 pmol of anti-Her2 Affibody-AlexaFluor680 via tail vein (with pre-injection background subtraction). Day 3 and 9 in vivo optical imaging signal was further correlated with ex vivo Her2 levels by western blot after sacrifice. Results Her2 expression decreased with 17-DMAG dose in vitro. In vivo optical imaging signal was reduced by 22.5% in Clone B (p=0.003) and by 9% in MCF7 parental tumors (p=0.23) at 3 days after 17-DMAG treatment; optical imaging signal recovered in both tumor types at day 6–9. In the carrier group no signal reduction was observed. Pearson correlation of in vivo optical imaging signal with ex vivo Her2 levels ranged from 0.73 to 0.89. Conclusion Optical imaging with an affibody can be used to non-invasively monitor changes in Her2 expression in vivo as a response to treatment with an Hsp90 inhibitor, with results similar to response measurements in PET imaging studies. PMID:22235098

  18. Discovery of RNA Binding Small Molecules Using Small Molecule Microarrays.

    PubMed

    Connelly, Colleen M; Abulwerdi, Fardokht A; Schneekloth, John S

    2017-01-01

    New methods to identify RNA-binding small molecules open yet unexplored opportunities for the pharmacological modulation of RNA-driven biology and disease states. One such approach is the use of small molecule microarrays (SMMs). Typically, SMMs are generated by spatially arraying and covalently linking a library of small molecules to a glass surface. Next, incubation of the arrays with a fluorescently labeled RNA reveals binding interactions that are detected upon slide imaging. The relative ease with which SMMs are manufactured enables the screening of multiple oligonucleotides in parallel against tens of thousands of small molecules, providing information about both binding and selectivity of identified RNA-small molecule interactions. This approach is useful for screening a broad variety of structurally and functionally diverse RNAs. Here, we present a general method for the preparation and use of SMMs to rapidly identify small molecules that selectively bind to an RNA of interest.

  19. Cooperative Ligand Binding to Linear Chain Molecules

    ERIC Educational Resources Information Center

    Applequist, Jon

    1977-01-01

    Summarizes the Ising model of ligand binding as it applies to cooperative binding to long chain molecules. Also presents some illustrations which help to visualize the connection between the interaction parameters and the shape of the binding isotherm. (Author/MR)

  20. Hydrophobic fluorescent probes introduce artifacts into single molecule tracking experiments due to non-specific binding.

    PubMed

    Zanetti-Domingues, Laura C; Tynan, Christopher J; Rolfe, Daniel J; Clarke, David T; Martin-Fernandez, Marisa

    2013-01-01

    Single-molecule techniques are powerful tools to investigate the structure and dynamics of macromolecular complexes; however, data quality can suffer because of weak specific signal, background noise and dye bleaching and blinking. It is less well-known, but equally important, that non-specific binding of probe to substrates results in a large number of immobile fluorescent molecules, introducing significant artifacts in live cell experiments. Following from our previous work in which we investigated glass coating substrates and demonstrated that the main contribution to this non-specific probe adhesion comes from the dye, we carried out a systematic investigation of how different dye chemistries influence the behaviour of spectrally similar fluorescent probes. Single-molecule brightness, bleaching and probe mobility on the surface of live breast cancer cells cultured on a non-adhesive substrate were assessed for anti-EGFR affibody conjugates with 14 different dyes from 5 different manufacturers, belonging to 3 spectrally homogeneous bands (491 nm, 561 nm and 638 nm laser lines excitation). Our results indicate that, as well as influencing their photophysical properties, dye chemistry has a strong influence on the propensity of dye-protein conjugates to adhere non-specifically to the substrate. In particular, hydrophobicity has a strong influence on interactions with the substrate, with hydrophobic dyes showing much greater levels of binding. Crucially, high levels of non-specific substrate binding result in calculated diffusion coefficients significantly lower than the true values. We conclude that the physic-chemical properties of the dyes should be considered carefully when planning single-molecule experiments. Favourable dye characteristics such as photostability and brightness can be offset by the propensity of a conjugate for non-specific adhesion.

  1. Comparison of Two Site-Specifically 18F-Labeled Affibodies for PET Imaging of EGFR Positive Tumors

    PubMed Central

    2015-01-01

    The epidermal growth factor receptor (EGFR) serves as an attractive target for cancer molecular imaging and therapy. Our previous positron emission tomography (PET) studies showed that the EGFR-targeting affibody molecules 64Cu-DOTA-ZEGFR:1907 and 18F-FBEM-ZEGFR:1907 can discriminate between high and low EGFR-expression tumors and have the potential for patient selection for EGFR-targeted therapy. Compared with 64Cu, 18F may improve imaging of EGFR-expression and is more suitable for clinical application, but the labeling reaction of 18F-FBEM-ZEGFR:1907 requires a long synthesis time. The aim of the present study is to develop a new generation of 18F labeled affibody probes (Al18F-NOTA-ZEGFR:1907 and 18F-CBT-ZEGFR:1907) and to determine whether they are suitable agents for imaging of EGFR expression. The first approach consisted of conjugating ZEGFR:1907 with NOTA and radiolabeling with Al18F to produce Al18F-NOTA-ZEGFR:1907. In a second approach the prosthetic group 18F-labeled-2-cyanobenzothiazole (18F-CBT) was conjugated to Cys-ZEGFR:1907 to produce 18F-CBT-ZEGFR:1907. Binding affinity and specificity of Al18F-NOTA-ZEGFR:1907 and 18F-CBT-ZEGFR:1907 to EGFR were evaluated using A431 cells. Biodistribution and PET studies were conducted on mice bearing A431 xenografts after injection of Al18F-NOTA-ZEGFR:1907 or 18F-CBT-ZEGFR:1907 with or without coinjection of unlabeled affibody proteins. The radiosyntheses of Al18F-NOTA-ZEGFR:1907 and 18F-CBT-ZEGFR:1907 were completed successfully within 40 and 120 min with a decay-corrected yield of 15% and 41% using a 2-step, 1-pot reaction and 2-step, 2-pot reaction, respectively. Both probes bound to EGFR with low nanomolar affinity in A431 cells. Although 18F-CBT-ZEGFR:1907 showed instability in vivo, biodistribution studies revealed rapid and high tumor accumulation and quick clearance from normal tissues except the bones. In contrast, Al18F-NOTA-ZEGFR:1907 demonstrated high in vitro and in vivo stability, high tumor uptake

  2. Feasibility of Affibody-Based Bioorthogonal Chemistry-Mediated Radionuclide Pretargeting.

    PubMed

    Altai, Mohamed; Perols, Anna; Tsourma, Maria; Mitran, Bogdan; Honarvar, Hadis; Robillard, Marc; Rossin, Raffaella; ten Hoeve, Wolter; Lubberink, Mark; Orlova, Anna; Karlström, Amelie Eriksson; Tolmachev, Vladimir

    2016-03-01

    Affibody molecules constitute a new class of probes for radionuclide tumor targeting. The small size of Affibody molecules is favorable for rapid localization in tumors and clearance from circulation. However, high renal reabsorption of Affibody molecules prevents the use of residualizing radiometals, including several promising low-energy β- and α-emitters, for radionuclide therapy. We tested a hypothesis that Affibody-based pretargeting mediated by a bioorthogonal interaction between trans-cyclooctene (TCO) and tetrazine would provide higher accumulation of radiometals in tumor xenografts than in the kidneys. TCO was conjugated to the anti-human epidermal growth factor receptor 2 (HER2) Affibody molecule Z2395. DOTA-tetrazine was labeled with (111)In and (177)Lu. In vitro pretargeting was studied in HER2-expressing SKOV-3 and BT474 cell lines. In vivo studies were performed on BALB/C nu/nu mice bearing SKOV-3 xenografts. (125)I-Z2395-TCO bound specifically to HER2-expressing cells in vitro with an affinity of 45 ± 16 pM. (111)In-tetrazine bound specifically and selectively to Z2395-TCO pretreated cells. In vivo studies demonstrated HER2-specific (125)I-Z2395-TCO accumulation in xenografts. TCO-mediated (111)In-tetrazine localization was shown in tumors, when the radiolabeled tracer was injected 4 h after an injection of Z2395-TCO. At 1 h after injection, the tumor uptake of (111)In-tetrazine and (177)Lu-tetrazine was approximately 2-fold higher than the renal uptake. Pretargeting provided more than a 56-fold reduction of renal uptake of (111)In in comparison with direct targeting. The feasibility of Affibody-based bioorthogonal chemistry-mediated pretargeting was demonstrated. The use of pretargeting provides a substantial reduction of radiometal accumulation in kidneys, creating preconditions for palliative radionuclide therapy. © 2016 by the Society of Nuclear Medicine and Molecular Imaging, Inc.

  3. Nanobubble-Affibody: Novel ultrasound contrast agents for targeted molecular ultrasound imaging of tumor.

    PubMed

    Yang, Hengli; Cai, Wenbin; Xu, Lei; Lv, Xiuhua; Qiao, Youbei; Li, Pan; Wu, Hong; Yang, Yilin; Zhang, Li; Duan, Yunyou

    2015-01-01

    Nanobubbles (NBs), as novel ultrasound contrast agents (UCAs), have attracted increasing attention in the field of molecular ultrasound imaging for tumors. However, the preparation of uniform-sized NBs is considered to be controversial, and poor tumor selectivity in in vivo imaging has been reported. In this study, we fabricated uniform nano-sized NBs (478.2 ± 29.7 nm with polydispersity index of 0.164 ± 0.044, n = 3) using a thin-film hydration method by controlling the thickness of phospholipid films; we then conjugated the NBs with Affibody molecules to produce nano-sized UCAs referred to as NB-Affibody with specific affinity to human epidermal growth factor receptor type 2 (HER2)-overexpressing tumors. NB-Affibody presented good ultrasound enhancement, demonstrating a peak intensity of 104.5 ± 2.1 dB under ultrasound contrast scanning. Ex vivo experiments further confirmed that the NB-Affibody conjugates were capable of targeting HER2-expressing tumor cells in vivo with high affinity. The newly prepared nano-sized NB-Affibody conjugates were observed to be novel targeted UCAs for efficient and safe specific molecular imaging and may have potential applications in early cancer quantitative diagnosis and targeted therapy in the future. Copyright © 2014 Elsevier Ltd. All rights reserved.

  4. Single molecule junction conductance and binding geometry

    NASA Astrophysics Data System (ADS)

    Kamenetska, Maria

    This Thesis addresses the fundamental problem of controlling transport through a metal-organic interface by studying electronic and mechanical properties of single organic molecule-metal junctions. Using a Scanning Tunneling Microscope (STM) we image, probe energy-level alignment and perform STM-based break junction (BJ) measurements on molecules bound to a gold surface. Using Scanning Tunneling Microscope-based break-junction (STM-BJ) techniques, we explore the effect of binding geometry on single-molecule conductance by varying the structure of the molecules, metal-molecule binding chemistry and by applying sub-nanometer manipulation control to the junction. These experiments are performed both in ambient conditions and in ultra high vacuum (UHV) at cryogenic temperatures. First, using STM imaging and scanning tunneling spectroscopy (STS) measurements we explore binding configurations and electronic properties of an amine-terminated benzene derivative on gold. We find that details of metal-molecule binding affect energy-level alignment at the interface. Next, using the STM-BJ technique, we form and rupture metal-molecule-metal junctions ˜104 times to obtain conductance-vs-extension curves and extract most likely conductance values for each molecule. With these measurements, we demonstrated that the control of junction conductance is possible through a choice of metal-molecule binding chemistry and sub-nanometer positioning. First, we show that molecules terminated with amines, sulfides and phosphines bind selectively on gold and therefore demonstrate constant conductance levels even as the junction is elongated and the metal-molecule attachment point is modified. Such well-defined conductance is also obtained with paracyclophane molecules which bind to gold directly through the pi system. Next, we are able to create metal-molecule-metal junctions with more than one reproducible conductance signatures that can be accessed by changing junction geometry. In the

  5. Anion binding to the ubiquitin molecule.

    PubMed Central

    Makhatadze, G. I.; Lopez, M. M.; Richardson, J. M.; Thomas, S. T.

    1998-01-01

    Effects of different salts (NaCl, MgCl2, CaCl2, GdmCl, NaBr, NaClO4, NaH2PO4, Na2SO4) on the stability of the ubiquitin molecule at pH 2.0 have been studied by differential scanning calorimetry, circular dichroism, and Tyr fluorescence spectroscopies. It is shown that all of the salts studied significantly increase the thermostability of the ubiquitin molecule, and that this stabilization can be interpreted in terms of anion binding. Estimated thermodynamic parameters of binding for Cl- show that this binding is relatively weak (Kd = 0.15 M) and is characterized by a negative enthalpy of -15 kJ/mol per site. Particularly surprising was the observed stabilizing effect of GdmCl through the entire concentration range studied (0.01-2 M), however, to a lesser extent than stabilization by NaCl. This stabilizing effect of GdmCl appears to arise from the binding of Cl- ions. Analysis of the observed changes in the stability of the ubiquitin molecule in the presence of GdmCl can be adequately described by combining the thermodynamic model of denaturant binding with Cl- binding effects. PMID:9541401

  6. Specific targeting of HER2-positive head and neck squamous cell carcinoma line HN5 by Idarubicin-ZHER2 affibody conjugate.

    PubMed

    Ghanemi, Marzieh; Pourshohod, Aminollah; Ghaffari, Mohammad Ali; Kheirollah, Alireza; Amin, Mansour; Zeinali, Majid; Jamalan, Mostafa

    2017-04-26

    Expression of human epidermal growth factor receptor type 2 (HER2) in head and neck squamous cell carcinoma (HNSCC) cell line HN5 can be employed with great opportunities of success for specific targeting of anti-cancer chemotherapeutic agents. In the current study, HER2-specific affibody molecule, ZHER2:342 (an engineered protein with great affinity for HER2 receptors) was selected for conjugation to idarubicin (an anti-neoplastic antibiotic). ZHER2:342 affibody gene with one added cysteine code at the its 5' end was synthesized de novo and then inserted into pET302 plasmid and transferred to E. Coli BL21 hosting system. After induction of protein expression, the recombinant ZHER2 affibody molecules were purified using Ni-NTA resin and purity was analyzed through SDS-PAGE. Affinity-purified affibody molecules were conjugated to idarubicin through a heterobifunctional crosslinker, sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (Sulfo-SMCC). Specific toxicity of idarubicin-ZHER2 affibody conjugate against two HER2-positive cells, HN5 and MCF7 was assessed through MTT assay after an exposure time of 48 hours with different concentrations of conjugate. Idarubicin in the non-conjugated form showed potent toxic effects against both cell lines, while HN5 cells were significantly more sensitive compared to MCF-7 cells. Dimeric ZHER2 affibody showed a mild decreasing effect on growth of both HN5 and MCF-7 cells at optimum concentration. Idarubicin-ZHER2 affibody conjugate at an optimum concentration reduced viability of HN5 cell line more efficiently compared to MCF-7 cell line. In conclusion, idarubicin-ZHER2 affibody conjugate in optimum concentrations can be used for specific targeting and killing of HN5 cells. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  7. Influence of molecular design on biodistribution and targeting properties of an Affibody-fused HER2-recognising anticancer toxin.

    PubMed

    Altai, Mohamed; Liu, Hao; Orlova, Anna; Tolmachev, Vladimir; Gräslund, Torbjörn

    2016-09-01

    Targeted delivery of toxins is a promising way to treat disseminated cancer. The use of monoclonal antibodies as targeting moiety has provided proof-of-principle for this approach. However, extravasation and tissue penetration rates of antibody-based immunotoxins are limited due to antibody bulkiness. The use of a novel class of targeting probes, Affibody molecules, provides smaller toxin-conjugated constructs, which may improve targeting. Earlier, we have demonstrated that affitoxins containing a HER2-targeting Affibody moiety and a deimmunized and truncated exotoxin A from Pseudomonas aeruginosa, PE38X8, provide highly selective toxicity to HER2-expressing cancer cells. To evaluate the influence of molecular design on targeting and biodistribution properties, a series of novel affitoxins were labelled with the residualizing radionuclide 111In. In this study, we have shown that the novel conjugates are more rapidly internalized compared with the parental affitoxin. The use of a (HE)3 purification tag instead of a hexahistidine tag enabled significant (p<0.05) reduction of the hepatic uptake of the affitoxin in a murine model. Fusion of the affitoxin with an albumin-binding domain (ABD) caused appreciable extension of the residence time in circulation and several-fold reduction of the renal uptake. The best variant, 111In-(HE)3-ZHER2-ABD-PE38X8, demonstrated receptor-specific accumulation in HER2-expressing SKOV-3 xenografts. In conclusion, a careful molecular design of scaffold protein based anticancer targeted toxins can appreciably improve their biodistribution and targeting properties.

  8. Biophysical characterization of DNA binding from single molecule force measurements

    PubMed Central

    Chaurasiya, Kathy R.; Paramanathan, Thayaparan; McCauley, Micah J.; Williams, Mark C.

    2010-01-01

    Single molecule force spectroscopy is a powerful method that uses the mechanical properties of DNA to explore DNA interactions. Here we describe how DNA stretching experiments quantitatively characterize the DNA binding of small molecules and proteins. Small molecules exhibit diverse DNA binding modes, including binding into the major and minor grooves and intercalation between base pairs of double-stranded DNA (dsDNA). Histones bind and package dsDNA, while other nuclear proteins such as high mobility group proteins bind to the backbone and bend dsDNA. Single-stranded DNA (ssDNA) binding proteins slide along dsDNA to locate and stabilize ssDNA during replication. Other proteins exhibit binding to both dsDNA and ssDNA. Nucleic acid chaperone proteins can switch rapidly between dsDNA and ssDNA binding modes, while DNA polymerases bind both forms of DNA with high affinity at distinct binding sites at the replication fork. Single molecule force measurements quantitatively characterize these DNA binding mechanisms, elucidating small molecule interactions and protein function. PMID:20576476

  9. Biophysical characterization of DNA binding from single molecule force measurements

    NASA Astrophysics Data System (ADS)

    Chaurasiya, Kathy R.; Paramanathan, Thayaparan; McCauley, Micah J.; Williams, Mark C.

    2010-09-01

    Single molecule force spectroscopy is a powerful method that uses the mechanical properties of DNA to explore DNA interactions. Here we describe how DNA stretching experiments quantitatively characterize the DNA binding of small molecules and proteins. Small molecules exhibit diverse DNA binding modes, including binding into the major and minor grooves and intercalation between base pairs of double-stranded DNA (dsDNA). Histones bind and package dsDNA, while other nuclear proteins such as high mobility group proteins bind to the backbone and bend dsDNA. Single-stranded DNA (ssDNA) binding proteins slide along dsDNA to locate and stabilize ssDNA during replication. Other proteins exhibit binding to both dsDNA and ssDNA. Nucleic acid chaperone proteins can switch rapidly between dsDNA and ssDNA binding modes, while DNA polymerases bind both forms of DNA with high affinity at distinct binding sites at the replication fork. Single molecule force measurements quantitatively characterize these DNA binding mechanisms, elucidating small molecule interactions and protein function.

  10. DNA-binding small molecules as inhibitors of transcription factors.

    PubMed

    Leung, Chung-Hang; Chan, Daniel Shiu-Hin; Ma, Victor Pui-Yan; Ma, Dik-Lung

    2013-07-01

    Accumulating evidence implicating the role of aberrant transcription factor signaling in the pathogenesis of various human diseases such as cancer and inflammation has stimulated the development of small molecule ligands capable of targeting transcription factor activity and modulating gene expression. The use of DNA-binding small molecules to selectively inhibit transcription factor-DNA interactions represents one possible approach toward this goal. In this review, we summarize the development of DNA-binding small molecule inhibitors of transcription factors from 2004 to 2011, and their binding mode and therapeutic potential will be discussed. © 2012 Wiley Periodicals, Inc.

  11. Free enthalpies of replacing water molecules in protein binding pockets.

    PubMed

    Riniker, Sereina; Barandun, Luzi J; Diederich, François; Krämer, Oliver; Steffen, Andreas; van Gunsteren, Wilfred F

    2012-12-01

    Water molecules in the binding pocket of a protein and their role in ligand binding have increasingly raised interest in recent years. Displacement of such water molecules by ligand atoms can be either favourable or unfavourable for ligand binding depending on the change in free enthalpy. In this study, we investigate the displacement of water molecules by an apolar probe in the binding pocket of two proteins, cyclin-dependent kinase 2 and tRNA-guanine transglycosylase, using the method of enveloping distribution sampling (EDS) to obtain free enthalpy differences. In both cases, a ligand core is placed inside the respective pocket and the remaining water molecules are converted to apolar probes, both individually and in pairs. The free enthalpy difference between a water molecule and a CH(3) group at the same location in the pocket in comparison to their presence in bulk solution calculated from EDS molecular dynamics simulations corresponds to the binding free enthalpy of CH(3) at this location. From the free enthalpy difference and the enthalpy difference, the entropic contribution of the displacement can be obtained too. The overlay of the resulting occupancy volumes of the water molecules with crystal structures of analogous ligands shows qualitative correlation between experimentally measured inhibition constants and the calculated free enthalpy differences. Thus, such an EDS analysis of the water molecules in the binding pocket may give valuable insight for potency optimization in drug design.

  12. Free enthalpies of replacing water molecules in protein binding pockets

    NASA Astrophysics Data System (ADS)

    Riniker, Sereina; Barandun, Luzi J.; Diederich, François; Krämer, Oliver; Steffen, Andreas; van Gunsteren, Wilfred F.

    2012-12-01

    Water molecules in the binding pocket of a protein and their role in ligand binding have increasingly raised interest in recent years. Displacement of such water molecules by ligand atoms can be either favourable or unfavourable for ligand binding depending on the change in free enthalpy. In this study, we investigate the displacement of water molecules by an apolar probe in the binding pocket of two proteins, cyclin-dependent kinase 2 and tRNA-guanine transglycosylase, using the method of enveloping distribution sampling (EDS) to obtain free enthalpy differences. In both cases, a ligand core is placed inside the respective pocket and the remaining water molecules are converted to apolar probes, both individually and in pairs. The free enthalpy difference between a water molecule and a CH3 group at the same location in the pocket in comparison to their presence in bulk solution calculated from EDS molecular dynamics simulations corresponds to the binding free enthalpy of CH3 at this location. From the free enthalpy difference and the enthalpy difference, the entropic contribution of the displacement can be obtained too. The overlay of the resulting occupancy volumes of the water molecules with crystal structures of analogous ligands shows qualitative correlation between experimentally measured inhibition constants and the calculated free enthalpy differences. Thus, such an EDS analysis of the water molecules in the binding pocket may give valuable insight for potency optimization in drug design.

  13. Affibody-mediated PET imaging of HER3 expression in malignant tumours

    PubMed Central

    Rosestedt, Maria; Andersson, Ken G.; Mitran, Bogdan; Tolmachev, Vladimir; Löfblom, John; Orlova, Anna; Ståhl, Stefan

    2015-01-01

    Human epidermal growth factor receptor 3 (HER3) is involved in the progression of various cancers and in resistance to therapies targeting the HER family. In vivo imaging of HER3 expression would enable patient stratification for anti-HER3 immunotherapy. Key challenges with HER3-targeting are the relatively low expression in HER3-positive tumours and HER3 expression in normal tissues. The use of positron-emission tomography (PET) provides advantages of high resolution, sensitivity and quantification accuracy compared to SPECT. Affibody molecules, imaging probes based on a non-immunoglobulin scaffold, provide high imaging contrast shortly after injection. The aim of this study was to evaluate feasibility of PET imaging of HER3 expression using 68Ga-labeled affibody molecules. The anti-HER3 affibody molecule HEHEHE-Z08698-NOTA was successfully labelled with 68Ga with high yield, purity and stability. The agent bound specifically to HER3-expressing cancer cells in vitro and in vivo. At 3 h pi, uptake of 68Ga-HEHEHE-Z08698-NOTA was significantly higher in xenografts with high HER3 expression (BT474, BxPC-3) than in xenografts with low HER3 expression (A431). In xenografts with high expression, tumour-to-blood ratios were >20, tumour-to-muscle >15, and tumour-to-bone >7. HER3-positive xenografts were visualised using microPET 3 h pi. In conclusion, PET imaging of HER3 expression is feasible using 68Ga-HEHEHE-Z08698-NOTA shortly after administration. PMID:26477646

  14. Single-molecule binding experiments on long time scales.

    PubMed

    Elenko, Mark P; Szostak, Jack W; van Oijen, Antoine M

    2010-08-01

    We describe an approach for performing single-molecule binding experiments on time scales from hours to days, allowing for the observation of slower kinetics than have been previously investigated by single-molecule techniques. Total internal reflection fluorescence microscopy is used to image the binding of labeled ligand to molecules specifically coupled to the surface of an optically transparent flow cell. Long-duration experiments are enabled by ensuring sufficient positional, chemical, thermal, and image stability. Principal components of this experimental stability include illumination timing, solution replacement, and chemical treatment of solution to reduce photodamage and photobleaching; and autofocusing to correct for spatial drift.

  15. EGFR-directed Affibody for fluorescence-guided glioma surgery: time-dose analysis (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Ribeiro de Souza, Ana Luiza; Marra, Kayla; Gunn, Jason R.; Elliott, Jonathan T.; Samkoe, Kimberley S.; Paulsen, Keith D.; Draney, Daniel R.; Feldwisch, Joachim

    2016-03-01

    The key to fluorescence guided surgical oncology is the ability to create specific contrast between normal and glioma tissue. The blood brain barrier that limits the delivery of substances to the normal brain is broken in tumors, allowing accumulation of agents in the tumor interior. However, for a clinical success, imaging agents should be in the infiltrative edges to minimize the resection of normal brain while enable the removal of tumor. The aberrant overexpression and/or activation of EGFR is associated with many types of cancers, including glioblastoma and the injection of a fluorescent molecule targeted to these receptors would improve tumor contrast during fluorescence guided surgery. Affibody molecules have intentional medium affinity and high potential specificity, which are the desirable features of a good surgical imaging agent. The aim of this study was evaluate the brain/glioma uptake of ABY029 labeled with near-infrared dye IRDye800CW after intravenous injection. Rats were either inoculated with orthotopic implantations of U251 human glioma cell line or PBS (shams control) in the brain. The tumors were allowed to grow for 2-3 weeks before carrying out fluorescent tracer experiments. Fluorescent imaging of ex vivo brain slices from rats was acquired at different time points after infection of fluorescently labeled EGFR-specific affibody to verify which time provided maximal contrast tumor to normal brain. Although the tumor was most clearly visualized after 1h of IRDye800CW-labeled ABY029 injection, the tumor location could be identified from the background after 48h. These results suggest that the NIR-labeled affibody examined shows excellent potential to increase surgical visualization for confirmed EGFR positive tumors.

  16. Design and In Vitro Evaluation of a Cytotoxic Conjugate Based on the Anti-HER2 Affibody Fused to the Fc Fragment of IgG1.

    PubMed

    Sochaj-Gregorczyk, Alicja M; Ludzia, Patryk; Kozdrowska, Emilia; Jakimowicz, Piotr; Sokolowska-Wedzina, Aleksandra; Otlewski, Jacek

    2017-08-03

    In our previous work we demonstrated that a small protein called affibody can be used for a cytotoxic conjugate development. The anti-HER2 affibody was armed with one moiety of a highly potent auristatin E and specifically killed HER2-positive cancer cells with a nanomolar IC50. The aim of this study was to improve the anti-HER2 affibody conjugate by increasing its size and the number of conjugated auristatin molecules. The affibody was fused to the Fc fragment of IgG1 resulting in a dimeric construct with the molecular weight of 68 kDa, referred to as ZHER2:2891-Fc, ensuring its prolonged half-life in the blood. Due to the presence of four interchain cysteines, the fusion protein could carry four drug molecules. Notably, the in vitro tests of the improved anti-HER2 conjugate revealed that it exhibits the IC50 of 130 pM for the HER2-positive SK-BR-3 cells and 98 nM for the HER2-negative MDA-MB-231 cells. High efficacy and specificity of the auristatin conjugate based on ZHER2:2891-Fc indicate that this construct is suitable for further in vivo evaluation.

  17. Design and In Vitro Evaluation of a Cytotoxic Conjugate Based on the Anti-HER2 Affibody Fused to the Fc Fragment of IgG1

    PubMed Central

    Sochaj-Gregorczyk, Alicja M.; Ludzia, Patryk; Kozdrowska, Emilia; Jakimowicz, Piotr; Sokolowska-Wedzina, Aleksandra; Otlewski, Jacek

    2017-01-01

    In our previous work we demonstrated that a small protein called affibody can be used for a cytotoxic conjugate development. The anti-HER2 affibody was armed with one moiety of a highly potent auristatin E and specifically killed HER2-positive cancer cells with a nanomolar IC50. The aim of this study was to improve the anti-HER2 affibody conjugate by increasing its size and the number of conjugated auristatin molecules. The affibody was fused to the Fc fragment of IgG1 resulting in a dimeric construct with the molecular weight of 68 kDa, referred to as ZHER2:2891-Fc, ensuring its prolonged half-life in the blood. Due to the presence of four interchain cysteines, the fusion protein could carry four drug molecules. Notably, the in vitro tests of the improved anti-HER2 conjugate revealed that it exhibits the IC50 of 130 pM for the HER2-positive SK-BR-3 cells and 98 nM for the HER2-negative MDA-MB-231 cells. High efficacy and specificity of the auristatin conjugate based on ZHER2:2891-Fc indicate that this construct is suitable for further in vivo evaluation. PMID:28771178

  18. Fluorescence-activated cell sorting of specific affibody-displaying staphylococci.

    PubMed

    Wernérus, Henrik; Samuelson, Patrik; Ståhl, Stefan

    2003-09-01

    Efficient enrichment of staphylococcal cells displaying specific heterologous affinity ligands on their cell surfaces was demonstrated by using fluorescence-activated cell sorting. Using bacterial surface display of peptide or protein libraries for the purpose of combinatorial protein engineering has previously been investigated by using gram-negative bacteria. Here, the potential for using a gram-positive bacterium was evaluated by employing the well-established surface expression system for Staphylococcus carnosus. Staphylococcus aureus protein A domains with binding specificity to immunoglobulin G or engineered specificity for the G protein of human respiratory syncytial virus were expressed as surface display on S. carnosus cells. The surface accessibility and retained binding specificity of expressed proteins were demonstrated in whole-cell enzyme and flow cytometry assays. Also, affibody-expressing target cells could be sorted essentially quantitatively from a moderate excess of background cells in a single step by using a high-stringency sorting mode. Furthermore, in a simulated library selection experiment, a more-than-25,000-fold enrichment of target cells could be achieved through only two rounds of cell sorting and regrowth. The results obtained indicate that staphylococcal surface display of affibody libraries combined with fluoresence-activated cell sorting might indeed constitute an attractive alternative to existing technology platforms for affinity-based selections.

  19. Fluorescence-Activated Cell Sorting of Specific Affibody-Displaying Staphylococci

    PubMed Central

    Wernérus, Henrik; Samuelson, Patrik; Ståhl, Stefan

    2003-01-01

    Efficient enrichment of staphylococcal cells displaying specific heterologous affinity ligands on their cell surfaces was demonstrated by using fluorescence-activated cell sorting. Using bacterial surface display of peptide or protein libraries for the purpose of combinatorial protein engineering has previously been investigated by using gram-negative bacteria. Here, the potential for using a gram-positive bacterium was evaluated by employing the well-established surface expression system for Staphylococcus carnosus. Staphylococcus aureus protein A domains with binding specificity to immunoglobulin G or engineered specificity for the G protein of human respiratory syncytial virus were expressed as surface display on S. carnosus cells. The surface accessibility and retained binding specificity of expressed proteins were demonstrated in whole-cell enzyme and flow cytometry assays. Also, affibody-expressing target cells could be sorted essentially quantitatively from a moderate excess of background cells in a single step by using a high-stringency sorting mode. Furthermore, in a simulated library selection experiment, a more-than-25,000-fold enrichment of target cells could be achieved through only two rounds of cell sorting and regrowth. The results obtained indicate that staphylococcal surface display of affibody libraries combined with fluoresence-activated cell sorting might indeed constitute an attractive alternative to existing technology platforms for affinity-based selections. PMID:12957920

  20. Small-Molecule Binding Aptamers: Selection Strategies, Characterization, and Applications

    NASA Astrophysics Data System (ADS)

    Ruscito, Annamaria; DeRosa, Maria

    2016-05-01

    Aptamers are single-stranded, synthetic oligonucleotides that fold into 3-dimensional shapes capable of binding non-covalently with high affinity and specificity to a target molecule. They are generated via an in vitro process known as the Systematic Evolution of Ligands by EXponential enrichment, from which candidates are screened and characterized, and then applied in aptamer-based biosensors for target detection. Aptamers for small molecule targets such as toxins, antibiotics, molecular markers, drugs, and heavy metals will be the focus of this review. Their accurate detection is ultimately needed for the protection and wellbeing of humans and animals. However, issues such as the drastic difference in size of the aptamer and small molecule make it challenging to select, characterize, and apply aptamers for the detection of small molecules. Thus, recent (since 2012) notable advances in small molecule aptamers, which have overcome some of these challenges, are presented here, while defining challenges that still exist are discussed

  1. Mapping small molecule binding data to structural domains

    PubMed Central

    2012-01-01

    Background Large-scale bioactivity/SAR Open Data has recently become available, and this has allowed new analyses and approaches to be developed to help address the productivity and translational gaps of current drug discovery. One of the current limitations of these data is the relative sparsity of reported interactions per protein target, and complexities in establishing clear relationships between bioactivity and targets using bioinformatics tools. We detail in this paper the indexing of targets by the structural domains that bind (or are likely to bind) the ligand within a full-length protein. Specifically, we present a simple heuristic to map small molecule binding to Pfam domains. This profiling can be applied to all proteins within a genome to give some indications of the potential pharmacological modulation and regulation of all proteins. Results In this implementation of our heuristic, ligand binding to protein targets from the ChEMBL database was mapped to structural domains as defined by profiles contained within the Pfam-A database. Our mapping suggests that the majority of assay targets within the current version of the ChEMBL database bind ligands through a small number of highly prevalent domains, and conversely the majority of Pfam domains sampled by our data play no currently established role in ligand binding. Validation studies, carried out firstly against Uniprot entries with expert binding-site annotation and secondly against entries in the wwPDB repository of crystallographic protein structures, demonstrate that our simple heuristic maps ligand binding to the correct domain in about 90 percent of all assessed cases. Using the mappings obtained with our heuristic, we have assembled ligand sets associated with each Pfam domain. Conclusions Small molecule binding has been mapped to Pfam-A domains of protein targets in the ChEMBL bioactivity database. The result of this mapping is an enriched annotation of small molecule bioactivity data and a

  2. RNA binding protein and binding site useful for expression of recombinant molecules

    DOEpatents

    Mayfield, Stephen

    2000-01-01

    The present invention relates to a gene expression system in eukaryotic and prokaryotic cells, preferably plant cells and intact plants. In particular, the invention relates to an expression system having a RB47 binding site upstream of a translation initiation site for regulation of translation mediated by binding of RB47 protein, a member of the poly(A) binding protein family. Regulation is further effected by RB60, a protein disulfide isomerase. The expression system is capable of functioning in the nuclear/cytoplasm of cells and in the chloroplast of plants. Translation regulation of a desired molecule is enhanced approximately 100 fold over that obtained without RB47 binding site activation.

  3. RNA binding protein and binding site useful for expression of recombinant molecules

    DOEpatents

    Mayfield, Stephen P.

    2006-10-17

    The present invention relates to a gene expression system in eukaryotic and prokaryotic cells, preferably plant cells and intact plants. In particular, the invention relates to an expression system having a RB47 binding site upstream of a translation initiation site for regulation of translation mediated by binding of RB47 protein, a member of the poly(A) binding protein family. Regulation is further effected by RB60, a protein disulfide isomerase. The expression system is capable of functioning in the nuclear/cytoplasm of cells and in the chloroplast of plants. Translation regulation of a desired molecule is enhanced approximately 100 fold over that obtained without RB47 binding site activation.

  4. Disordered Binding of Small Molecules to Aβ(12–28)*

    PubMed Central

    Convertino, Marino; Vitalis, Andreas; Caflisch, Amedeo

    2011-01-01

    In recent years, an increasing number of small molecules and short peptides have been identified that interfere with aggregation and/or oligomerization of the Alzheimer β-amyloid peptide (Aβ). Many of them possess aromatic moieties, suggesting a dominant role for those in interacting with Aβ along various stages of the aggregation process. In this study, we attempt to elucidate whether interactions of such aromatic inhibitors with monomeric Aβ(12–28) point to a common mechanism of action by performing atomistic molecular dynamics simulations at equilibrium. Our results suggest that, independently of the presence of inhibitors, monomeric Aβ(12–28) populates a partially collapsed ensemble that is largely devoid of canonical secondary structure at 300 K and neutral pH. The small molecules have different affinities for Aβ(12–28) that can be partially rationalized by the balance of aromatic and charged moieties constituting the molecules. There are no predominant binding modes, although aggregation inhibitors preferentially interact with the N-terminal portion of the fragment (residues 13–20). Analysis of the free energy landscape of Aβ(12–28) reveals differences highlighted by altered populations of a looplike conformer in the presence of inhibitors. We conclude that intrinsic disorder of Aβ persists at the level of binding small molecules and that inhibitors can significantly alter properties of monomeric Aβ via multiple routes of differing specificity. PMID:21969380

  5. Small-molecule ligands of methyl-lysine binding proteins.

    PubMed

    Herold, J Martin; Wigle, Tim J; Norris, Jacqueline L; Lam, Robert; Korboukh, Victoria K; Gao, Cen; Ingerman, Lindsey A; Kireev, Dmitri B; Senisterra, Guillermo; Vedadi, Masoud; Tripathy, Ashutosh; Brown, Peter J; Arrowsmith, Cheryl H; Jin, Jian; Janzen, William P; Frye, Stephen V

    2011-04-14

    Proteins which bind methylated lysines ("readers" of the histone code) are important components in the epigenetic regulation of gene expression and can also modulate other proteins that contain methyl-lysine such as p53 and Rb. Recognition of methyl-lysine marks by MBT domains leads to compaction of chromatin and a repressed transcriptional state. Antagonists of MBT domains would serve as probes to interrogate the functional role of these proteins and initiate the chemical biology of methyl-lysine readers as a target class. Small-molecule MBT antagonists were designed based on the structure of histone peptide-MBT complexes and their interaction with MBT domains determined using a chemiluminescent assay and ITC. The ligands discovered antagonize native histone peptide binding, exhibiting 5-fold stronger binding affinity to L3MBTL1 than its preferred histone peptide. The first cocrystal structure of a small molecule bound to L3MBTL1 was determined and provides new insights into binding requirements for further ligand design.

  6. Staphylococcal enterotoxins bind H-2Db molecules on macrophages

    NASA Technical Reports Server (NTRS)

    Beharka, A. A.; Iandolo, J. J.; Chapes, S. K.; Spooner, B. S. (Principal Investigator)

    1995-01-01

    We screened a panel of monoclonal antibodies against selected macrophage cell surface molecules for their ability to inhibit enterotoxin binding to major histocompatibility complex class II-negative C2D (H-2b) macrophages. Two monoclonal antibodies, HB36 and TIB126, that are specific for the alpha 2 domain of major histocompatibility complex class I, blocked staphylococcal enterotoxins A and B (SEA and SEB, respectively) binding to C2D macrophages in a specific and concentration-dependent manner. Inhibitory activities were haplotype-specific in that SEA and SEB binding to H-2k or H-2d macrophages was not inhibited by either monoclonal antibody. HB36, but not TIB126, inhibited enterotoxin-induced secretion of cytokines by H-2b macrophages. Lastly, passive protection of D-galactosamine-sensitized C2D mice by injection with HB36 antibody prevented SEB-induced death. Therefore, SEA and SEB binding to the alpha 2 domain of the H-2Db molecule induces biological activity and has physiological consequences.

  7. Small Molecule Ligands of Methyl-Lysine Binding Proteins

    PubMed Central

    Herold, J. Martin; Wigle, Tim J.; Norris, Jacqueline L.; Lam, Robert; Korboukh, Victoria K.; Gao, Cen; Ingerman, Lindsey A.; Kireev, Dmitri B.; Senisterra, Guillermo; Vedadi, Masoud; Tripathy, Ashutosh; Brown, Peter J.; Arrowsmith, Cheryl H.; Jin, Jian; Janzen, William P.; Frye, Stephen V.

    2011-01-01

    Proteins which bind methylated lysines (“readers” of the histone code) are important components in the epigenetic regulation of gene expression and can also modulate other proteins that contain methyl-lysine such as p53 and Rb. Recognition of methyl-lysine marks by MBT domains leads to compaction of chromatin and a repressed transcriptional state. Antagonists of MBT domains would serve as probes to interrogate the functional role of these proteins and initiate the chemical biology of methyl-lysine readers as a target class. Small molecule MBT antagonists were designed based on the structure of histone peptide-MBT complexes and their interaction with MBT domains determined using a chemiluminescent assay and ITC. The ligands discovered antagonize native histone peptide binding, exhibiting 5-fold stronger binding affinity to L3MBTL1 than its preferred histone peptide. The first co-crystal structure of a small molecule bound to L3MBTL1 was determined and provides new insights into binding requirements for further ligand design. PMID:21417280

  8. Staphylococcal enterotoxins bind H-2Db molecules on macrophages

    NASA Technical Reports Server (NTRS)

    Beharka, A. A.; Iandolo, J. J.; Chapes, S. K.; Spooner, B. S. (Principal Investigator)

    1995-01-01

    We screened a panel of monoclonal antibodies against selected macrophage cell surface molecules for their ability to inhibit enterotoxin binding to major histocompatibility complex class II-negative C2D (H-2b) macrophages. Two monoclonal antibodies, HB36 and TIB126, that are specific for the alpha 2 domain of major histocompatibility complex class I, blocked staphylococcal enterotoxins A and B (SEA and SEB, respectively) binding to C2D macrophages in a specific and concentration-dependent manner. Inhibitory activities were haplotype-specific in that SEA and SEB binding to H-2k or H-2d macrophages was not inhibited by either monoclonal antibody. HB36, but not TIB126, inhibited enterotoxin-induced secretion of cytokines by H-2b macrophages. Lastly, passive protection of D-galactosamine-sensitized C2D mice by injection with HB36 antibody prevented SEB-induced death. Therefore, SEA and SEB binding to the alpha 2 domain of the H-2Db molecule induces biological activity and has physiological consequences.

  9. 188Re-ZHER2:V2, a promising affibody-based targeting agent against HER2-expressing tumors: preclinical assessment.

    PubMed

    Altai, Mohamed; Wållberg, Helena; Honarvar, Hadis; Strand, Joanna; Orlova, Anna; Varasteh, Zohreh; Sandström, Mattias; Löfblom, John; Larsson, Erik; Strand, Sven-Erik; Lubberink, Mark; Ståhl, Stefan; Tolmachev, Vladimir

    2014-11-01

    Affibody molecules are small (7 kDa) nonimmunoglobulin scaffold proteins with favorable tumor-targeting properties. Studies concerning the influence of chelators on biodistribution of (99m)Tc-labeled Affibody molecules demonstrated that the variant with a C-terminal glycyl-glycyl-glycyl-cysteine peptide-based chelator (designated ZHER2:V2) has the best biodistribution profile in vivo and the lowest renal retention of radioactivity. The aim of this study was to evaluate (188)Re-ZHER2:V2 as a potential candidate for radionuclide therapy of human epidermal growth factor receptor type 2 (HER2)-expressing tumors. ZHER2:V2 was labeled with (188)Re using a gluconate-containing kit. Targeting of HER2-overexpressing SKOV-3 ovarian carcinoma xenografts in nude mice was studied for a dosimetry assessment. Binding of (188)Re-ZHER2:V2 to living SKOV-3 cells was demonstrated to be specific, with an affinity of 6.4 ± 0.4 pM. The biodistribution study showed a rapid blood clearance (1.4 ± 0.1 percentage injected activity per gram [%ID/g] at 1 h after injection). The tumor uptake was 14 ± 2, 12 ± 2, 5 ± 2, and 1.8 ± 0.5 %IA/g at 1, 4, 24, and 48 h after injection, respectively. The in vivo targeting of HER2-expressing xenografts was specific. Already at 4 h after injection, tumor uptake exceeded kidney uptake (2.1 ± 0.2 %IA/g). Scintillation-camera imaging showed that tumor xenografts were the only sites with prominent accumulation of radioactivity at 4 h after injection. Based on the biokinetics, a dosimetry evaluation for humans suggests that (188)Re-ZHER2:V2 would provide an absorbed dose to tumor of 79 Gy without exceeding absorbed doses of 23 Gy to kidneys and 2 Gy to bone marrow. This indicates that future human radiotherapy studies may be feasible. (188)Re-ZHER2:V2 can deliver high absorbed doses to tumors without exceeding kidney and bone marrow toxicity limits. © 2014 by the Society of Nuclear Medicine and Molecular Imaging, Inc.

  10. An affibody-adalimumab hybrid blocks combined IL-6 and TNF-triggered serum amyloid A secretion in vivo.

    PubMed

    Yu, Feifan; Gudmundsdotter, Lindvi; Akal, Anastassja; Gunneriusson, Elin; Frejd, Fredrik; Nygren, Per-Åke

    2014-01-01

    In inflammatory disease conditions, the regulation of the cytokine system is impaired, leading to tissue damages. Here, we used protein engineering to develop biologicals suitable for blocking a combination of inflammation driving cytokines by a single construct. From a set of interleukin (IL)-6-binding affibody molecules selected by phage display, five variants with a capability of blocking the interaction between complexes of soluble IL-6 receptor α (sIL-6Rα) and IL-6 and the co-receptor gp130 were identified. In cell assays designed to analyze any blocking capacity of the classical or the alternative (trans) signaling IL-6 pathways, one variant, ZIL-6_13 with an affinity (KD) for IL-6 of ∼500 pM, showed the best performance. To construct fusion proteins ("AffiMabs") with dual cytokine specificities, ZIL-6_13 was fused to either the N- or C-terminus of both the heavy and light chains of the anti-tumor necrosis factor (TNF) monoclonal antibody adalimumab (Humira®). One AffiMab construct with ZIL-6_13 positioned at the N-terminus of the heavy chain, denoted ZIL-6_13-HCAda, was determined to be the most optimal, and it was subsequently evaluated in an acute Serum Amyloid A (SAA) model in mice. Administration of the AffiMab or adalimumab prior to challenge with a mix of IL-6 and TNF reduced the levels of serum SAA in a dose-dependent manner. Interestingly, the highest dose (70 mg/kg body weight) of adalimumab only resulted in a 50% reduction of SAA-levels, whereas the corresponding dose of the ZIL-6_13-HCAda AffiMab with combined IL-6/TNF specificity, resulted in SAA levels below the detection limit.

  11. Kinetics of small molecule inhibitor binding to p38 kinase.

    PubMed

    Thurmond, R L; Wadsworth, S A; Schafer, P H; Zivin, R A; Siekierka, J J

    2001-11-01

    p38 mitogen-activated protein kinase (MAPK) (p38/p38-alpha/CSBP2/RK) has been implicated in the regulation of many proinflammatory pathways. Because of this, it has received much attention as a potential drug target for controlling diseases such as rheumatoid arthritis, endotoxic shock, inflammatory bowel disease, osteoporosis, and many others. A number of small molecule inhibitors of this kinase have been described, and in this paper we have used surface plasmon resonance to directly measure and quantitate their binding to p38. Despite the relatively low molecular mass (approximately 400 Da) of these inhibitors, specific binding can be observed. For the two most potent inhibitors studied, SB 203580 and RWJ 67657, dissociation constants, K(d)'s, of 22 and 10 nm, respectively, were obtained. These values closely match the IC(5)0 values observed in a cell-based TNF alpha release assay implying that p38 plays a major role in TNF alpha release. The association and dissociation rates for the binding of these inhibitors to p38 have also been quantitated. SB 203580 and RWJ 67657 have very similar association rates of around 8 x 10(5) m(-1) x s(-1), and the differences in affinity are determined by different dissociation rates. The weaker binding compounds have dissociation rates similar to SB 203580, but the association rates vary by an order of magnitude or more. The direct measurement of compounds binding to p38 may help in understanding the difference between potency and efficacy for these inhibitors. This in turn may yield clues on how to develop better inhibitors.

  12. Small-Molecule Binding Aptamers: Selection Strategies, Characterization, and Applications

    PubMed Central

    Ruscito, Annamaria; DeRosa, Maria C.

    2016-01-01

    Aptamers are single-stranded, synthetic oligonucleotides that fold into 3-dimensional shapes capable of binding non-covalently with high affinity and specificity to a target molecule. They are generated via an in vitro process known as the Systematic Evolution of Ligands by EXponential enrichment, from which candidates are screened and characterized, and then used in various applications. These applications range from therapeutic uses to biosensors for target detection. Aptamers for small molecule targets such as toxins, antibiotics, molecular markers, drugs, and heavy metals will be the focus of this review. Their accurate detection is needed for the protection and wellbeing of humans and animals. However, the small molecular weights of these targets, including the drastic size difference between the target and the oligonucleotides, make it challenging to select, characterize, and apply aptamers for their detection. Thus, recent (since 2012) notable advances in small molecule aptamers, which have overcome some of these challenges, are presented here, while defining challenges that still exist are discussed. PMID:27242994

  13. Phosphate binding energy and catalysis by small and large molecules.

    PubMed

    Morrow, Janet R; Amyes, Tina L; Richard, John P

    2008-04-01

    Catalysis is an important process in chemistry and enzymology. The rate acceleration for any catalyzed reaction is the difference between the activation barriers for the uncatalyzed (Delta G(HO)(#)) and catalyzed (Delta G(Me)(#)) reactions, which corresponds to the binding energy (Delta G(S)(#) = Delta G(Me)(#)-Delta G(HO)(#)) for transfer of the reaction transition state from solution to the catalyst. This transition state binding energy is a fundamental descriptor of catalyzed reactions, and its evaluation is necessary for an understanding of any and all catalytic processes. We have evaluated the transition state binding energies obtained from interactions between low molecular weight metal ion complexes or high molecular weight protein catalysts and the phosphate group of bound substrate. Work on catalysis by small molecules is exemplified by studies on the mechanism of action of Zn2(1)(H2O). A binding energy of Delta G(S)(#) = -9.6 kcal/mol was determined for Zn2(1)(H2O)-catalyzed cleavage of the RNA analogue HpPNP. The pH-rate profile for this cleavage reaction showed that there is optimal catalytic activity at high pH, where the catalyst is in the basic form [Zn2(1)(HO-)]. However, it was also shown that the active form of the catalyst is Zn2(1)(H2O) and that this recognizes the C2-oxygen-ionized substrate in the cleavage reaction. The active catalyst Zn2(1)(H2O) shows a high affinity for oxyphosphorane transition state dianions and a stable methyl phosphate transition state analogue, compared with the affinity for phosphate monoanion substrates. The transition state binding energies, Delta G(S)(#), for cleavage of HpPNP catalyzed by a variety of Zn2+ and Eu3+ metal ion complexes reflect the increase in the catalytic activity with increasing total positive charge at the catalyst. These values of Delta G(S)(#) are affected by interactions between the metal ion and its ligands, but these effects are small in comparison with Delta G(S)(#) observed for catalysis

  14. Zooming into the binding groove of HLA molecules: which positions and which substitutions change peptide binding most?

    PubMed

    van Deutekom, Hanneke W M; Keşmir, Can

    2015-08-01

    Human leukocyte antigen (HLA) genes are the most polymorphic genes in the human genome. Almost all polymorphic residues are located in the peptide-binding groove, resulting in different peptide-binding preferences. Whether a single amino acid change can alter the peptide-binding repertoire of an HLA molecule has never been shown. To experimentally quantify the contribution of a single amino acid change to the peptide repertoire of even a single HLA molecule requires an immense number of HLA peptide-binding measurements. Therefore, we used an in silico method to study the effect of single mutations on the peptide repertoires. We predicted the peptide-binding repertoire of a large set of HLA molecules and used the overlap of the peptide-binding repertoires of each pair of HLA molecules that differ on a single position to measure how much single substitutions change the peptide binding. We found that the effect of a single substitution in the peptide-binding groove depends on the substituted position and the amino acids involved. The positions that alter peptide binding most are the most polymorphic ones, while those that are hardly variable among HLA molecules have the lowest effect on the peptide repertoire. Although expected, the relationship between functional divergence and polymorphism of HLA molecules has never been shown before. Additionally, we show that a single substitution in HLA-B molecules has more effect on the peptide-binding repertoire compared to that in HLA-A molecules. This provides an (alternative) explanation for the larger polymorphism of HLA-B molecules compared to HLA-A molecules.

  15. Proteoform-specific protein binding of small molecules in complex matrices

    USDA-ARS?s Scientific Manuscript database

    Characterizing the specific binding between protein targets and small molecules is critically important for drug discovery. Conventional assays require isolation and purification of small molecules from complex matrices through multistep chromatographic fractionation, which may alter their original ...

  16. Single Molecule Kinetics of ENTH Binding to Lipid Membranes

    SciTech Connect

    Rozovsky, Sharon; Forstner, Martin B.; Sondermann, Holger; Groves, Jay T.

    2012-04-03

    Transient recruitment of proteins to membranes is a fundamental mechanism by which the cell exerts spatial and temporal control over proteins’ localization and interactions. Thus, the specificity and the kinetics of peripheral proteins’ membrane residence are an attribute of their function. In this article, we describe the membrane interactions of the interfacial epsin N-terminal homology (ENTH) domain with its target lipid phosphatidylinositol (4,5)-bisphosphate (PtdIns(4,5)P2). The direct visualization and quantification of interactions of single ENTH molecules with supported lipid bilayers is achieved using total internal reflection fluorescence microscopy (TIRFM) with a time resolution of 13 ms. This enables the recording of the kinetic behavior of ENTH interacting with membranes with physiologically relevant concentrations of PtdIns(4,5)P2 despite the low effective binding affinity. Subsequent single fluorophore tracking permits us to build up distributions of residence times and to measure ENTH dissociation rates as a function of membrane composition. In addition, due to the high time resolution, we are able to resolve details of the motion of ENTH associated with a simple, homogeneous membrane. In this case ENTH’s diffusive transport appears to be the result of at least three different diffusion processes.

  17. Selective binding of single-stranded DNA-binding proteins onto DNA molecules adsorbed on single-walled carbon nanotubes.

    PubMed

    Nii, Daisuke; Hayashida, Takuya; Yamaguchi, Yuuki; Ikawa, Shukuko; Shibata, Takehiko; Umemura, Kazuo

    2014-09-01

    Single-stranded DNA-binding (SSB) proteins were treated with hybrids of DNA and single-walled carbon nanotubes (SWNTs) to examine the biological function of the DNA molecules adsorbed on the SWNT surface. When single-stranded DNA (ssDNA) was used for the hybridization, significant binding of the SSB molecules to the ssDNA-SWNT hybrids was observed by using atomic force microscopy (AFM) and agarose gel electrophoresis. When double-stranded DNA (dsDNA) was used, the SSB molecules did not bind to the dsDNA-SWNT hybrids in most of the conditions that we evaluated. A specifically modified electrophoresis procedure was used to monitor the locations of the DNA, SSB, and SWNT molecules. Our results clearly showed that ssDNA/dsDNA molecules on the SWNT surfaces retained their single-stranded/double-stranded structures.

  18. Measuring HER2-Receptor Expression In Metastatic Breast Cancer Using [68Ga]ABY-025 Affibody PET/CT

    PubMed Central

    Sörensen, Jens; Velikyan, Irina; Sandberg, Dan; Wennborg, Anders; Feldwisch, Joachim; Tolmachev, Vladimir; Orlova, Anna; Sandström, Mattias; Lubberink, Mark; Olofsson, Helena; Carlsson, Jörgen; Lindman, Henrik

    2016-01-01

    Purpose: Positron Emission Tomography (PET) imaging of HER2 expression could potentially be used to select patients for HER2-targed therapy, predict response based on uptake and be used for monitoring. In this phase I/II study the HER2-binding Affibody molecule ABY-025 was labeled with 68Ga-gallium ([68Ga]ABY-025) for PET to study effect of peptide mass, test-retest variability and correlation of quantified uptake in tumors to histopathology. Experimental design: Sixteen women with known metastatic breast cancer and on-going treatment were included and underwent FDG PET/CT to identify viable metastases. After iv injection of 212±46 MBq [68Ga]ABY-025 whole-body PET was performed at 1, 2 and 4 h. In the first 10 patients (6 with HER2-positive and 4 with HER2-negative primary tumors), [68Ga]ABY-025 PET/CT with two different doses of injected peptide was performed one week apart. In the last six patients (5 HER2-positive and 1 HER2-negative primary tumors), repeated [68Ga]ABY-025 PET were performed one week apart as a test-retest of uptake in individual lesions. Biopsies from 16 metastases in 12 patients were collected for verification of HER2 expression by immunohistochemistry and in-situ hybridization. Results: Imaging 4h after injection with high peptide content discriminated HER2-positive metastases best (p<0.01). PET SUV correlated with biopsy HER2-scores (r=0.91, p<0.001). Uptake was five times higher in HER2-positive than in HER2-negative lesions with no overlap (p=0.005). The test-retest intra-class correlation was r=0.996. [68Ga]ABY-025 PET correctly identified conversion and mixed expression of HER2 and targeted treatment was changed in 3 of the 16 patients. Conclusion: [68Ga]ABY-025 PET accurately quantifies whole-body HER2-receptor status in metastatic breast cancer. PMID:26877784

  19. Visualization of Surface-tethered Large DNA Molecules with a Fluorescent Protein DNA Binding Peptide.

    PubMed

    Lee, Seonghyun; Jo, Kyubong

    2016-06-23

    Large DNA molecules tethered on the functionalized glass surface have been utilized in polymer physics and biochemistry particularly for investigating interactions between DNA and its binding proteins. Here, we report a method that uses fluorescent microscopy for visualizing large DNA molecules tethered on the surface. First, glass coverslips are biotinylated and passivated by coating with biotinylated polyethylene glycol, which specifically binds biotinylated DNA via avidin protein linkers and significantly reduces undesirable binding from non-specific interactions of proteins or DNA molecules on the surface. Second, the DNA molecules are biotinylated by two different methods depending on their terminals. The blunt ended DNA is tagged with biotinylated dUTP at its 3' hydroxyl terminus, by terminal transferase, while the sticky ended DNA is hybridized with biotinylated complimentary oligonucleotides by DNA ligase. Finally, a microfluidic shear flow makes single DNA molecules stretch to their full contour lengths after being stained with fluorescent protein-DNA binding peptide (FP-DBP).

  20. SpyLigase peptide–peptide ligation polymerizes affibodies to enhance magnetic cancer cell capture

    PubMed Central

    Fierer, Jacob O.; Veggiani, Gianluca; Howarth, Mark

    2014-01-01

    Individual proteins can now often be modified with atomic precision, but there are still major obstacles to connecting proteins into larger assemblies. To direct protein assembly, ideally, peptide tags would be used, providing the minimal perturbation to protein function. However, binding to peptides is generally weak, so assemblies are unstable over time and disassemble with force or harsh conditions. We have recently developed an irreversible protein–peptide interaction (SpyTag/SpyCatcher), based on a protein domain from Streptococcus pyogenes, that locks itself together via spontaneous isopeptide bond formation. Here we develop irreversible peptide–peptide interaction, through redesign of this domain and genetic dissection into three parts: a protein domain termed SpyLigase, which now ligates two peptide tags to each other. All components expressed efficiently in Escherichia coli and peptide tags were reactive at the N terminus, at the C terminus, or at internal sites. Peptide–peptide ligation enabled covalent and site-specific polymerization of affibodies or antibodies against the tumor markers epidermal growth factor receptor (EGFR) and HER2. Magnetic capture of circulating tumor cells (CTCs) is one of the most promising approaches to improve cancer prognosis and management, but CTC capture is limited by inefficient recovery of cells expressing low levels of tumor antigen. SpyLigase-assembled protein polymers made possible the isolation of cancerous cells expressing lower levels of tumor antigen and should have general application in enhancing molecular capture. PMID:24639550

  1. Ferritin nanocage with intrinsically disordered proteins and affibody: A platform for tumor targeting with extended pharmacokinetics.

    PubMed

    Lee, Na Kyeong; Lee, Eun Jung; Kim, Soyoun; Nam, Gi-Hoon; Kih, Minwoo; Hong, Yeonsun; Jeong, Cherlhyun; Yang, Yoosoo; Byun, Youngro; Kim, In-San

    2017-08-15

    Ferritin nanocages are of particular interest as a novel platform for drug and vaccine delivery, diagnosis, biomineralization scaffold and more, due to their perfect and complex symmetry, ideal physical properties, high biocompatibility, low toxicity profiles as well as easy manipulation by genetic or chemical strategies. However, a short half-life is still a hurdle for the translation of ferritin-based nanomedicines into the clinic. Here, we developed a series of rationally designed long circulating ferritin nanocages (LCFNs) with 'Intrinsically Disordered Proteins (IDP)' as a stealth layer for extending the half-life of ferritin nanocages. Through predictions with 3D modelling, the LCFNs were designed, generated and their pharmacokinetic parameters including half-life, clearance rate, mean residence time, and more, were evaluated by qualitative and quantitative analysis. LCFNs have a tenfold increased half-life and overall improved pharmacokinetic parameters compared to wild-type ferritin nanocages (wtFN), corresponding to the low binding against bone marrow-derived macrophages (BMDMs) and endothelial cells. Subsequently, a tumor targeting moiety, epidermal growth factor receptor (EGFR)-targeting affibody peptide, was fused to LCFNs for evaluating their potential as a theragnostic platform. The tumor targeting-LCFNs successfully accumulated to the tumor tissue, by efficient targeting via active and passive properties, and also the shielding effect of IDP in vivo. This strategy can be applied to other protein-based nanocages for further progressing their use in the field of nanomedicine. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. Chemocavity: specific concavity in protein reserved for the binding of biologically functional small molecules.

    PubMed

    Soga, Shinji; Shirai, Hiroki; Kobori, Masato; Hirayama, Noriaki

    2008-08-01

    The idea that there should be a specific site on a protein for a particular functional small molecule is widespread. It is, however, usually not so easy to understand what characteristics of the site determine the binding ability of the functional small molecule. We have focused on the concurrence rate of the 20 standard amino acids at such binding sites. In order to correlate the concurrence rate and the specific binding site, we have analyzed high-quality X-ray structures of complexes between proteins and small molecules. A novel index characterizing the binding site based on the concurrency rate has been introduced. Using this index we have identified that there is a specific concavity designated as a chemocavity where a specific group of small molecules, i.e., canonical molecular group, is highly inclined to be bound. This study has demonstrated that a chemocavity is reserved for a specific canonical molecular group, and the prevalent idea has been confirmed.

  3. Characterization of binding specificities of bovine leucocyte class I molecules: impacts for rational epitope discovery

    PubMed Central

    Hansen, Andreas M.; Rasmussen, Michael; Svitek, Nicholas; Harndahl, Mikkel; Golde, William T.; Barlow, John; Nene, Vishvanath; Buus, Søren

    2014-01-01

    The binding of peptides to classical major histocompatibility complex (MHC) class I proteins is the single most selective step in antigen presentation. However, the peptide-binding specificity of cattle MHC (bovine leucocyte antigen, BoLA) class I (BoLA-I) molecules remains poorly characterized. Here, we demonstrate how a combination of high-throughput assays using positional scanning combinatorial peptide libraries, peptide dissociation, and peptide-binding affinity binding measurements can be combined with bioinformatics to effectively characterize the functionality of BoLA-I molecules. Using this strategy, we characterized eight BoLA-I molecules, and found the peptide specificity to resemble that of human MHC-I molecules with primary anchors most often at P2 and P9, and occasional auxiliary P1/P3/P5/P6 anchors. We analyzed nine reported CTL epitopes from Theileria parva, and in eight cases, stable and high affinity binding was confirmed. A set of peptides were tested for binding affinity to the eight BoLA proteins and used to refine the predictors of peptide–MHC binding NetMHC and NetMHCpan. The inclusion of BoLA-specific peptide-binding data led to a significant improvement in prediction accuracy for reported T. parva CTL epitopes. For reported CTL epitopes with weak or no predicted binding, these refined prediction methods suggested presence of nested minimal epitopes with high-predicted binding affinity. The enhanced affinity of the alternative peptides was in all cases confirmed experimentally. This study demonstrates how biochemical high-throughput assays combined with immunoinformatics can be used to characterize the peptide-binding motifs of BoLA-I molecules, boosting performance of MHC peptide-binding prediction methods, and empowering rational epitope discovery in cattle. PMID:25186069

  4. Molecular level studies on binding modes of labeling molecules with polyalanine peptides

    NASA Astrophysics Data System (ADS)

    Mao, Xiaobo; Wang, Chenxuan; Ma, Xiaojing; Zhang, Min; Liu, Lei; Zhang, Lan; Niu, Lin; Zeng, Qindao; Yang, Yanlian; Wang, Chen

    2011-04-01

    In this work, the binding modes of typical labeling molecules (thioflavin T (ThT), Congo red (CR) and copper(ii) phthalocyanine tetrasulfonic acid tetrasodium salt (PcCu(SO3Na)4)) on pentaalanine, which is a model peptide segment of amyloidpeptides, have been resolved at the molecular level by using scanning tunneling microscopy (STM). In the STM images, ThT molecules are predominantly adsorbed parallel to the peptide strands and two binding modes could be identified. It was found that ThT molecules are preferentially binding on top of the peptide strand, and the mode of intercalated between neighboring peptides also exists. The parallel binding mode of CR molecules can be observed with pentaalaninepeptides. Besides the binding modes of labeling molecules, the CR and PcCu(SO3Na)4 display different adsorption affinity with the pentaalaninepeptides. The results could be beneficial for obtaining molecular level insight of the interactions between labeling molecules and peptides.In this work, the binding modes of typical labeling molecules (thioflavin T (ThT), Congo red (CR) and copper(ii) phthalocyanine tetrasulfonic acid tetrasodium salt (PcCu(SO3Na)4)) on pentaalanine, which is a model peptide segment of amyloidpeptides, have been resolved at the molecular level by using scanning tunneling microscopy (STM). In the STM images, ThT molecules are predominantly adsorbed parallel to the peptide strands and two binding modes could be identified. It was found that ThT molecules are preferentially binding on top of the peptide strand, and the mode of intercalated between neighboring peptides also exists. The parallel binding mode of CR molecules can be observed with pentaalaninepeptides. Besides the binding modes of labeling molecules, the CR and PcCu(SO3Na)4 display different adsorption affinity with the pentaalaninepeptides. The results could be beneficial for obtaining molecular level insight of the interactions between labeling molecules and peptides. Electronic

  5. Alternative peptide binding motifs of Qa-2 class Ib molecules define rules for binding of self and nonself peptides.

    PubMed

    Tabaczewski, P; Chiang, E; Henson, M; Stroynowski, I

    1997-09-15

    Studies of naturally processed peptides eluted from membrane-bound and soluble isoforms of murine class Ib Qa-2 molecules determined several features of these ligands, such as the conserved nonameric length and the preferred usage of specific residues at four to six of nine peptide positions. The structural information derived from these studies proved insufficient to distinguish between two interpretations: 1) that Qa-2 are peptide receptors of higher stringency than ordinary class I molecules, and 2) that Qa-2 molecules, like classical class I Ags, bind diverse arrays of peptides. We have addressed this issue by a systematic analysis of peptide residues involved in the binding of membrane-bound Qa-2 molecule, MQ9b. The optimal binding of synthetic peptides in vitro occurs at neutral pH. Two dominant anchors are required for peptide binding to MQ9b: His at position 7 and a hydrophobic residue, Leu, Ile, or Phe, at position 9. In addition, one or two auxiliary anchors participate in binding. The identity and the position of the auxiliary anchors differ from peptide to peptide, suggesting that the binding motifs defined from pool sequencing are composed of many superimposed alternative motifs present in individual peptides. The number of anchors used by Qa-2 peptides is similar to that found in ligands of classical class I Ags. Consequently, the Qa-2 are predicted to bind large repertoires of self and nonself peptides. In support of this interpretation we demonstrate that MQ9b binds strongly 5 of 17 motif-positive, pathogen-derived synthetic peptides.

  6. Nanopore Detector based analysis of single-molecule conformational kinetics and binding interactions

    PubMed Central

    Winters-Hilt, Stephen

    2006-01-01

    Background A Nanopore Detector provides a means to transduce single molecule events into observable channel current changes. Nanopore-based detection can report directly, or indirectly, on single molecule kinetics. The nanopore-based detector can directly measure molecular characteristics in terms of the blockade properties of individual molecules – this is possible due to the kinetic information that is embedded in the blockade measurements, where the adsorption-desorption history of the molecule to the surrounding channel, and the configurational changes in the molecule itself, imprint on the ionic flow through the channel. This rich source of information offers prospects for DNA sequencing and single nucleotide polymorphism (SNP) analysis. A nanopore-based detector can also measure molecular characteristics indirectly, by using a reporter molecule that binds to certain molecules, with subsequent distinctive blockade by the bound-molecule complex. Results It is hypothesized that reaction histories of individual molecules can be observed on model DNA/DNA, DNA/Protein, and Protein/Protein systems. Preliminary results are all consistent with this hypothesis. Nanopore detection capabilities are also described for highly discriminatory biosensing, binding strength characterization, and rapid immunological screening. Conclusion In essence, the heart of chemistry is now accessible to a new, single-molecule, observation method that can track both external molecular binding states, and internal conformation states. PMID:17118143

  7. Prediction of small molecule binding property of protein domains with Bayesian classifiers based on Markov chains.

    PubMed

    Bulashevska, Alla; Stein, Martin; Jackson, David; Eils, Roland

    2009-12-01

    Accurate computational methods that can help to predict biological function of a protein from its sequence are of great interest to research biologists and pharmaceutical companies. One approach to assume the function of proteins is to predict the interactions between proteins and other molecules. In this work, we propose a machine learning method that uses a primary sequence of a domain to predict its propensity for interaction with small molecules. By curating the Pfam database with respect to the small molecule binding ability of its component domains, we have constructed a dataset of small molecule binding and non-binding domains. This dataset was then used as training set to learn a Bayesian classifier, which should distinguish members of each class. The domain sequences of both classes are modelled with Markov chains. In a Jack-knife test, our classification procedure achieved the predictive accuracies of 77.2% and 66.7% for binding and non-binding classes respectively. We demonstrate the applicability of our classifier by using it to identify previously unknown small molecule binding domains. Our predictions are available as supplementary material and can provide very useful information to drug discovery specialists. Given the ubiquitous and essential role small molecules play in biological processes, our method is important for identifying pharmaceutically relevant components of complete proteomes. The software is available from the author upon request.

  8. Latrophilins Function as Heterophilic Cell-adhesion Molecules by Binding to Teneurins

    PubMed Central

    Boucard, Antony A.; Maxeiner, Stephan; Südhof, Thomas C.

    2014-01-01

    Latrophilin-1, -2, and -3 are adhesion-type G protein-coupled receptors that are auxiliary α-latrotoxin receptors, suggesting that they may have a synaptic function. Using pulldowns, we here identify teneurins, type II transmembrane proteins that are also candidate synaptic cell-adhesion molecules, as interactors for the lectin-like domain of latrophilins. We show that teneurin binds to latrophilins with nanomolar affinity and that this binding mediates cell adhesion, consistent with a role of teneurin binding to latrophilins in trans-synaptic interactions. All latrophilins are subject to alternative splicing at an N-terminal site; in latrophilin-1, this alternative splicing modulates teneurin binding but has no effect on binding of latrophilin-1 to another ligand, FLRT3. Addition to cultured neurons of soluble teneurin-binding fragments of latrophilin-1 decreased synapse density, suggesting that latrophilin binding to teneurin may directly or indirectly influence synapse formation and/or maintenance. These observations are potentially intriguing in view of the proposed role for Drosophila teneurins in determining synapse specificity. However, teneurins in Drosophila were suggested to act as homophilic cell-adhesion molecules, whereas our findings suggest a heterophilic interaction mechanism. Thus, we tested whether mammalian teneurins also are homophilic cell-adhesion molecules, in addition to binding to latrophilins as heterophilic cell-adhesion molecules. Strikingly, we find that although teneurins bind to each other in solution, homophilic teneurin-teneurin binding is unable to support stable cell adhesion, different from heterophilic teneurin-latrophilin binding. Thus, mammalian teneurins act as heterophilic cell-adhesion molecules that may be involved in trans-neuronal interaction processes such as synapse formation or maintenance. PMID:24273166

  9. Characterization of binding specificities of Bovine Leucocyte class I molecules: Impacts for rational epitope discovery

    USDA-ARS?s Scientific Manuscript database

    The binding of peptides to classical major histocompatibility complex (MHC) class-I proteins is the single most selective step in antigen presentation. However, the peptide binding specificity of cattle MHC (bovine leucocyte antigen, BoLA) class I (BoLA-I) molecules remains poorly characterized. Her...

  10. Small Molecule Microarrays Enable the Identification of a Selective, Quadruplex-Binding Inhibitor of MYC Expression.

    PubMed

    Felsenstein, Kenneth M; Saunders, Lindsey B; Simmons, John K; Leon, Elena; Calabrese, David R; Zhang, Shuling; Michalowski, Aleksandra; Gareiss, Peter; Mock, Beverly A; Schneekloth, John S

    2016-01-15

    The transcription factor MYC plays a pivotal role in cancer initiation, progression, and maintenance. However, it has proven difficult to develop small molecule inhibitors of MYC. One attractive route to pharmacological inhibition of MYC has been the prevention of its expression through small molecule-mediated stabilization of the G-quadruplex (G4) present in its promoter. Although molecules that bind globally to quadruplex DNA and influence gene expression are well-known, the identification of new chemical scaffolds that selectively modulate G4-driven genes remains a challenge. Here, we report an approach for the identification of G4-binding small molecules using small molecule microarrays (SMMs). We use the SMM screening platform to identify a novel G4-binding small molecule that inhibits MYC expression in cell models, with minimal impact on the expression of other G4-associated genes. Surface plasmon resonance (SPR) and thermal melt assays demonstrated that this molecule binds reversibly to the MYC G4 with single digit micromolar affinity, and with weaker or no measurable binding to other G4s. Biochemical and cell-based assays demonstrated that the compound effectively silenced MYC transcription and translation via a G4-dependent mechanism of action. The compound induced G1 arrest and was selectively toxic to MYC-driven cancer cell lines containing the G4 in the promoter but had minimal effects in peripheral blood mononucleocytes or a cell line lacking the G4 in its MYC promoter. As a measure of selectivity, gene expression analysis and qPCR experiments demonstrated that MYC and several MYC target genes were downregulated upon treatment with this compound, while the expression of several other G4-driven genes was not affected. In addition to providing a novel chemical scaffold that modulates MYC expression through G4 binding, this work suggests that the SMM screening approach may be broadly useful as an approach for the identification of new G4-binding small

  11. Investigation of the binding modes between AIE-active molecules and dsDNA by single molecule force spectroscopy

    NASA Astrophysics Data System (ADS)

    Chen, Ying; Ma, Ke; Hu, Ting; Jiang, Bo; Xu, Bin; Tian, Wenjing; Sun, Jing Zhi; Zhang, Wenke

    2015-05-01

    AIE (aggregation-induced emission)-active molecules hold promise for the labeling of biomolecules as well as living cells. The study of the binding modes of such molecules to biomolecules, such as nucleic acids and proteins, will shed light on a deeper understanding of the mechanisms of molecular interactions and eventually facilitate the design/preparation of new AIE-active bioprobes. Herein, we studied the binding modes of double-stranded DNA (dsDNA) with two types of synthetic AIE-active molecules, namely, tetraphenylethene-derived dicationic compounds (cis-TPEDPy and trans-TPEDPy) and anthracene-derived dicationic compounds (DSAI and DSABr-C6) using single molecule force spectroscopy (SMFS) and circular dichroism (CD) spectroscopy. The experimental data indicate that DSAI can strongly intercalate into DNA base pairs, while DSABr-C6 is unable to intercalate into DNA due to the steric hindrance of the alkyl side chains. Cis-TPEDPy and trans-TPEDPy can also intercalate into DNA base pairs, but the binding shows strong ionic strength dependence. Multiple binding modes of TPEDPy with dsDNA have been discussed. In addition, the electrostatic interaction enhanced intercalation of cis-TPEDPy with dsDNA has also been revealed.AIE (aggregation-induced emission)-active molecules hold promise for the labeling of biomolecules as well as living cells. The study of the binding modes of such molecules to biomolecules, such as nucleic acids and proteins, will shed light on a deeper understanding of the mechanisms of molecular interactions and eventually facilitate the design/preparation of new AIE-active bioprobes. Herein, we studied the binding modes of double-stranded DNA (dsDNA) with two types of synthetic AIE-active molecules, namely, tetraphenylethene-derived dicationic compounds (cis-TPEDPy and trans-TPEDPy) and anthracene-derived dicationic compounds (DSAI and DSABr-C6) using single molecule force spectroscopy (SMFS) and circular dichroism (CD) spectroscopy. The

  12. Small molecules that allosterically inhibit p21-activated kinase activity by binding to the regulatory p21-binding domain.

    PubMed

    Kim, Duk-Joong; Choi, Chang-Ki; Lee, Chan-Soo; Park, Mee-Hee; Tian, Xizhe; Kim, Nam Doo; Lee, Kee-In; Choi, Joong-Kwon; Ahn, Jin Hee; Shin, Eun-Young; Shin, Injae; Kim, Eung-Gook

    2016-04-29

    p21-activated kinases (PAKs) are key regulators of actin dynamics, cell proliferation and cell survival. Deregulation of PAK activity contributes to the pathogenesis of various human diseases, including cancer and neurological disorders. Using an ELISA-based screening protocol, we identified naphtho(hydro)quinone-based small molecules that allosterically inhibit PAK activity. These molecules interfere with the interactions between the p21-binding domain (PBD) of PAK1 and Rho GTPases by binding to the PBD. Importantly, they inhibit the activity of full-length PAKs and are selective for PAK1 and PAK3 in vitro and in living cells. These compounds may potentially be useful for determining the details of the PAK signaling pathway and may also be used as lead molecules in the development of more selective and potent PAK inhibitors.

  13. Guiding the design of synthetic DNA-binding molecules with massively parallel sequencing.

    PubMed

    Meier, Jordan L; Yu, Abigail S; Korf, Ian; Segal, David J; Dervan, Peter B

    2012-10-24

    Genomic applications of DNA-binding molecules require an unbiased knowledge of their high affinity sites. We report the high-throughput analysis of pyrrole-imidazole polyamide DNA-binding specificity in a 10(12)-member DNA sequence library using affinity purification coupled with massively parallel sequencing. We find that even within this broad context, the canonical pairing rules are remarkably predictive of polyamide DNA-binding specificity. However, this approach also allows identification of unanticipated high affinity DNA-binding sites in the reverse orientation for polyamides containing β/Im pairs. These insights allow the redesign of hairpin polyamides with different turn units capable of distinguishing 5'-WCGCGW-3' from 5'-WGCGCW-3'. Overall, this study displays the power of high-throughput methods to aid the optimal targeting of sequence-specific minor groove binding molecules, an essential underpinning for biological and nanotechnological applications.

  14. The boundary molecules in a lysozyme pattern exhibit preferential antibody binding.

    PubMed

    Gao, Pei; Cai, Yuguang

    2008-09-16

    Lysozyme was immobilized on a prefabricated carboxylic acid terminated chemical template, forming a tightly packed, one monolayer thick lysozyme pattern. Polyclonal anti-lysozyme antibodies can bind to the immobilized lysozyme pattern. Atomic force microscope (AFM) observation reveals that the antibodies bind to the lysozyme molecules on the pattern edge before they bind to the lysozyme molecules in the pattern interior. Better spatial accessibility and flexibility of the lysozyme molecules on the pattern edge are used to explain the observed antibody binding preference. The topographies of the lysozyme pattern also affect the antibody binding. The antibodies bind to the edge lysozyme from the top if the lysozyme pattern is half-buried in a 10 A deep channel, whereas the antibodies bind to the edge lysozyme from the side if the lysozyme pattern is immobilized on a protruding terrace. The observed "edge effect" suggests that, for the same protein coverage, reducing the protein pattern feature to the nanoscale will improve the overall binding activity of the immobilized protein toward the antibody.

  15. The role of water molecules in stereoselectivity of glucose/galactose-binding protein

    NASA Astrophysics Data System (ADS)

    Kim, Minsup; Cho, Art E.

    2016-11-01

    Using molecular dynamics (MD) simulation methods, we attempted to explain the experimental results on ligand specificity of glucose/galactose-binding protein (GGBP) to β-D-glucose and β-D-galactose. For the simulation, a three-dimensional structure of GGBP was prepared, and homology modeling was performed to generate variant structures of GGBP with mutations at Asp14. Then, docking was carried out to find a reasonable β-D-glucose and β-D-galactose binding conformations with GGBP. Subsequent molecular dynamics simulations of β-D-glucose–GGBP and β-D-galactose–GGBP complexes and estimation of the orientation and stability of water molecules at the binding site revealed how water molecules influence ligand specificity. In our simulation, water molecules mediated interactions of β-D-glucose or β-D-galactose with residue 14 of GGBP. In this mechanism, the Phe16Ala mutant leaves both sugar molecules free to move, and the specific role of water molecules were eliminated, while the wild type, Asp14Asn mutant, and Asp14Glu mutant make hydrogen bond interactions with β-D-glucose more favorable. Our results demonstrate that bound water molecules at the binding site of GGBP are related to localized conformational change, contributing to ligand specificity of GGBP for β-D-glucose over β-D-galactose.

  16. Energetics of displacing water molecules from protein binding sites: consequences for ligand optimization.

    PubMed

    Michel, Julien; Tirado-Rives, Julian; Jorgensen, William L

    2009-10-28

    A strategy in drug design is to consider enhancing the affinity of lead molecules with structural modifications that displace water molecules from a protein binding site. Because success of the approach is uncertain, clarification of the associated energetics was sought in cases where similar structural modifications yield qualitatively different outcomes. Specifically, free-energy perturbation calculations were carried out in the context of Monte Carlo statistical mechanics simulations to investigate ligand series that feature displacement of ordered water molecules in the binding sites of scytalone dehydratase, p38-alphaMAP kinase, and EGFR kinase. The change in affinity for a ligand modification is found to correlate with the ease of displacement of the ordered water molecule. However, as in the EGFR example, the binding affinity may diminish if the free-energy increase due to the removal of the bound water molecule is not more than compensated by the additional interactions of the water-displacing moiety. For accurate computation of the effects of ligand modifications, a complete thermodynamic analysis is shown to be needed. It requires identification of the location of water molecules in the protein-ligand interface and evaluation of the free-energy changes associated with their removal and with the introduction of the ligand modification. Direct modification of the ligand in free-energy calculations is likely to trap the ordered molecule and provide misleading guidance for lead optimization.

  17. Perlecan is recruited by dystroglycan to nodes of Ranvier and binds the clustering molecule gliomedin

    PubMed Central

    Colombelli, Cristina; Palmisano, Marilena; Eshed-Eisenbach, Yael; Zambroni, Desirée; Pavoni, Ernesto; Ferri, Cinzia; Saccucci, Stefania; Nicole, Sophie; Soininen, Raija; McKee, Karen K.; Yurchenco, Peter D.; Peles, Elior; Wrabetz, Lawrence

    2015-01-01

    Fast neural conduction requires accumulation of Na+ channels at nodes of Ranvier. Dedicated adhesion molecules on myelinating cells and axons govern node organization. Among those, specific laminins and dystroglycan complexes contribute to Na+ channel clustering at peripheral nodes by unknown mechanisms. We show that in addition to facing the basal lamina, dystroglycan is found near the nodal matrix around axons, binds matrix components, and participates in initial events of nodogenesis. We identify the dystroglycan-ligand perlecan as a novel nodal component and show that dystroglycan is required for the selective accumulation of perlecan at nodes. Perlecan binds the clustering molecule gliomedin and enhances clustering of node of Ranvier components. These data show that proteoglycans have specific roles in peripheral nodes and indicate that peripheral and central axons use similar strategies but different molecules to form nodes of Ranvier. Further, our data indicate that dystroglycan binds free matrix that is not organized in a basal lamina. PMID:25646087

  18. Possible role for cell-surface carbohydrate-binding molecules in lymphocyte recirculation

    PubMed Central

    1983-01-01

    We are investigating the hypothesis that carbohydrate-binding molecules on the cell surface are involved in the recirculation of lymphocytes from the bloodstream into lymphoid organs. This phenomenon requires the specific attachment of circulating lymphocytes to the endothelial cells of postcapillary venules. Using an in vitro assay to measure the adhesive interaction between lymphocytes and postcapillary venules, we have found that L-fucose, D mannose, and the L-fucose-rich, sulfated polysaccharide fucoidin specifically inhibit this binding interaction. L-fucose shows stereo-selective inhibitory activity at concentrations greater than 18 mM while fucoidin produces 50% inhibition at approximately 1-5 X 10(-8) M. Fucoidin appears to interact with the lymphocyte, and not the postcapillary venule, to inhibit binding. These data suggest that cell surface carbohydrates (fucoselike) and carbohydrate-binding molecules (cell surface lectins) may contribute to the specific attachment of lymphocytes to postcapillary venules. PMID:6833380

  19. Simple molecular model for the binding of antibiotic molecules to bacterial ion channels

    NASA Astrophysics Data System (ADS)

    Mafé, Salvador; Ramírez, Patricio; Alcaraz, Antonio

    2003-10-01

    A molecular model aimed at explaining recent experimental data by Nestorovich et al. [Proc. Natl. Acad. Sci. USA 99, 9789 (2002)] on the interaction of ampicillin molecules with the constriction zone in a channel of the general bacterial porin, OmpF (outer membrane protein F), is presented. The model extends T. L. Hill's theory for intermolecular interactions in a pair of binding sites [J. Am. Chem. Soc. 78, 3330 (1956)] by incorporating two binding ions and two pairs of interacting sites. The results provide new physical insights on the role of the complementary pattern of the charge distributions in the ampicillin molecule and the narrowest part of the channel pore. Charge matching of interacting sites facilitates drug binding. The dependence of the number of ampicillin binding events per second with the solution pH and salt concentration is explained qualitatively using a reduced number of fundamental concepts.

  20. Rapid and Accurate Prediction and Scoring of Water Molecules in Protein Binding Sites

    PubMed Central

    Ross, Gregory A.; Morris, Garrett M.; Biggin, Philip C.

    2012-01-01

    Water plays a critical role in ligand-protein interactions. However, it is still challenging to predict accurately not only where water molecules prefer to bind, but also which of those water molecules might be displaceable. The latter is often seen as a route to optimizing affinity of potential drug candidates. Using a protocol we call WaterDock, we show that the freely available AutoDock Vina tool can be used to predict accurately the binding sites of water molecules. WaterDock was validated using data from X-ray crystallography, neutron diffraction and molecular dynamics simulations and correctly predicted 97% of the water molecules in the test set. In addition, we combined data-mining, heuristic and machine learning techniques to develop probabilistic water molecule classifiers. When applied to WaterDock predictions in the Astex Diverse Set of protein ligand complexes, we could identify whether a water molecule was conserved or displaced to an accuracy of 75%. A second model predicted whether water molecules were displaced by polar groups or by non-polar groups to an accuracy of 80%. These results should prove useful for anyone wishing to undertake rational design of new compounds where the displacement of water molecules is being considered as a route to improved affinity. PMID:22396746

  1. Predicting the multi-modal binding propensity of small molecules: towards an understanding of drug promiscuity.

    PubMed

    Park, Keunwan; Lee, Soyoung; Ahn, Hee-Sung; Kim, Dongsup

    2009-08-01

    Drug promiscuity is one of the key issues in current drug development. Many famous drugs have turned out to behave unexpectedly due to their propensity to bind to multiple targets. One of the primary reasons for this promiscuity is that drugs bind to multiple distinctive target environments, a feature that we call multi-modal binding. Accordingly, investigations into whether multi-modal binding propensities can be predicted, and if so, whether the features determining this behavior can be found, would be an important advance. In this study, we have developed a structure-based classifier that predicts whether small molecules will bind to multiple distinct binding sites. The binding sites for all ligands in the Protein Data Bank (PDB) were clustered by binding site similarity, and the ligands that bind to many dissimilar binding sites were identified as multi-modal binding ligands. The mono-binding ligands were also collected, and the classifiers were built using various machine-learning algorithms. A 10-fold cross-validation procedure showed 70-85% accuracy depending on the choice of machine-learning algorithm, and the different definitions used to identify multi-modal binding ligands. In addition, a quantified importance measurement for global and local descriptors was also provided, which suggests that the local features are more likely to have an effect on multi-modal binding than the global ones. The interpretable global and local descriptors were also ranked by their importance. To test the classifier on real examples, several test sets including well-known promiscuous drugs were collected by a literature and database search. Despite the difficulty in constructing appropriate testable sets, the classifier showed reasonable results that were consistent with existing information on drug behavior. Finally, a test on natural enzyme substrates and artificial drugs suggests that the natural compounds tend to exhibit a broader range of multi-modal binding than the

  2. MHC2NNZ: A novel peptide binding prediction approach for HLA DQ molecules

    NASA Astrophysics Data System (ADS)

    Xie, Jiang; Zeng, Xu; Lu, Dongfang; Liu, Zhixiang; Wang, Jiao

    2017-07-01

    The major histocompatibility complex class II (MHC-II) molecule plays a crucial role in immunology. Computational prediction of MHC-II binding peptides can help researchers understand the mechanism of immune systems and design vaccines. Most of the prediction algorithms for MHC-II to date have made large efforts in human leukocyte antigen (HLA, the name of MHC in Human) molecules encoded in the DR locus. However, HLA DQ molecules are equally important and have only been made less progress because it is more difficult to handle them experimentally. In this study, we propose an artificial neural network-based approach called MHC2NNZ to predict peptides binding to HLA DQ molecules. Unlike previous artificial neural network-based methods, MHC2NNZ not only considers sequence similarity features but also captures the chemical and physical properties, and a novel method incorporating these properties is proposed to represent peptide flanking regions (PFR). Furthermore, MHC2NNZ improves the prediction accuracy by combining with amino acid preference at more specific positions of the peptides binding core. By evaluating on 3549 peptides binding to six most frequent HLA DQ molecules, MHC2NNZ is demonstrated to outperform other state-of-the-art MHC-II prediction methods.

  3. Proteoform-Specific Protein Binding of Small Molecules in Complex Matrices.

    PubMed

    Gil, Geuncheol; Mao, Pan; Avula, Bharathi; Ali, Zulfiqar; Chittiboyina, Amar G; Khan, Ikhlas A; Walker, Larry A; Wang, Daojing

    2017-02-17

    Characterizing the specific binding between protein targets and small molecules is critically important for drug discovery. Conventional assays require isolation and purification of small molecules from complex matrices through multistep chromatographic fractionation, which may alter their original bioactivity. Most proteins undergo posttranslational modification, and only certain proteoforms have the right conformation with accessible domains and available residues for small molecule binding. We developed a top-down mass spectrometry (MS) centric workflow for rapid evaluation of the bioactivity of crude botanical extracts after a one-step reaction. Our assay distinguished covalent from noncovalent binding and mapped the residue for covalent binding between bioactive constituents and specific proteoforms of the target protein. We augmented our approach with a nanoflow liquid chromatography-selected reaction monitoring (SRM)-MS assay for simultaneous identification and label-free multiplex quantitation of small molecules in the crude botanical extracts. Our assay was validated for various proteoforms of human serum albumin, which plays a key role in pharmacokinetics of small molecules in vivo. We demonstrated the utility of our proteoform-specific assay for evaluating thymoquinone in crude botanical extracts, studying its pharmacokinetics in human blood, and interpreting its toxicity to human breast cancer cells in tissue culture.

  4. Binding energies of hydrogen molecules to isoreticular metal-organic framework materials.

    PubMed

    Sagara, Tatsuhiko; Klassen, James; Ortony, Julia; Ganz, Eric

    2005-07-01

    Recently, several novel isoreticular metal-organic framework (IRMOF) structures have been fabricated and tested for hydrogen storage applications. To improve our understanding of these materials, and to promote quantitative calculations and simulations, the binding energies of hydrogen molecules to the MOF have been studied. High-quality second-order Moller-Plesset (MP2) calculations using the resolution of the identity approximation and the quadruple zeta QZVPP basis set were used. These calculations use terminated molecular fragments from the MOF materials. For H2 on the zinc oxide corners, the MP2 binding energy using Zn4O(HCO2)6 molecule is 6.28 kJ/mol. For H2 on the linkers, the binding energy is calculated using lithium-terminated molecular fragments. The MP2 results with coupled-cluster singles and doubles and noniterative triples method corrections and charge-transfer corrections are 4.16 kJ/mol for IRMOF-1, 4.72 kJ/mol for IRMOF-3, 4.86 kJ/mol for IRMOF-6, 4.54 kJ/mol for IRMOF-8, 5.50 and 4.90 kJ/mol for IRMOF-12, 4.87 and 4.84 kJ/mol for IRMOF-14, 5.42 kJ/mol for IRMOF-18, and 4.97 and 4.66 kJ/mol for IRMOF-993. The larger linkers are all able to bind multiple hydrogen molecules per side. The linkers of IRMOF-12, IRMOF-993, and IRMOF-14 can bind two to three, three, and four hydrogen molecules per side, respectively. In general, the larger linkers have the largest binding energies, and, together with the enhanced surface area available for binding, will provide increased hydrogen storage. We also find that adding up NH2 or CH3 groups to each linker can provide up to a 33% increase in the binding energy.

  5. Binding energies of hydrogen molecules to isoreticular metal-organic framework materials

    NASA Astrophysics Data System (ADS)

    Sagara, Tatsuhiko; Klassen, James; Ortony, Julia; Ganz, Eric

    2005-07-01

    Recently, several novel isoreticular metal-organic framework (IRMOF) structures have been fabricated and tested for hydrogen storage applications. To improve our understanding of these materials, and to promote quantitative calculations and simulations, the binding energies of hydrogen molecules to the MOF have been studied. High-quality second-order Møller-Plesset (MP2) calculations using the resolution of the identity approximation and the quadruple zeta QZVPP basis set were used. These calculations use terminated molecular fragments from the MOF materials. For H2 on the zinc oxide corners, the MP2 binding energy using Zn4O(HCO2)6 molecule is 6.28kJ/mol. For H2 on the linkers, the binding energy is calculated using lithium-terminated molecular fragments. The MP2 results with coupled-cluster singles and doubles and noniterative triples method corrections and charge-transfer corrections are 4.16kJ/mol for IRMOF-1, 4.72kJ/mol for IRMOF-3, 4.86kJ/mol for IRMOF-6, 4.54kJ/mol for IRMOF-8, 5.50 and 4.90kJ/mol for IRMOF-12, 4.87 and 4.84kJ/mol for IRMOF-14, 5.42kJ/mol for IRMOF-18, and 4.97 and 4.66kJ/mol for IRMOF-993. The larger linkers are all able to bind multiple hydrogen molecules per side. The linkers of IRMOF-12, IRMOF-993, and IRMOF-14 can bind two to three, three, and four hydrogen molecules per side, respectively. In general, the larger linkers have the largest binding energies, and, together with the enhanced surface area available for binding, will provide increased hydrogen storage. We also find that adding up NH2 or CH3 groups to each linker can provide up to a 33% increase in the binding energy.

  6. Prediction of peptide binding to a major histocompatibility complex class I molecule based on docking simulation.

    PubMed

    Ishikawa, Takeshi

    2016-10-01

    Binding between major histocompatibility complex (MHC) class I molecules and immunogenic epitopes is one of the most important processes for cell-mediated immunity. Consequently, computational prediction of amino acid sequences of MHC class I binding peptides from a given sequence may lead to important biomedical advances. In this study, an efficient structure-based method for predicting peptide binding to MHC class I molecules was developed, in which the binding free energy of the peptide was evaluated by two individual docking simulations. An original penalty function and restriction of degrees of freedom were determined by analysis of 361 published X-ray structures of the complex and were then introduced into the docking simulations. To validate the method, calculations using a 50-amino acid sequence as a prediction target were performed. In 27 calculations, the binding free energy of the known peptide was within the top 5 of 166 peptides generated from the 50-amino acid sequence. Finally, demonstrative calculations using a whole sequence of a protein as a prediction target were performed. These data clearly demonstrate high potential of this method for predicting peptide binding to MHC class I molecules.

  7. Factors affecting the threading of axle molecules through macrocycles: Binding constants for semirotaxane formation

    PubMed Central

    Clifford, Thomas; Abushamleh, Ahmad; Busch, Daryle H.

    2002-01-01

    The threading of more or less linear axle molecules through macrocyclic molecules, a fundamental process relating to the formation of interlocked molecular structures, has been investigated through the study in acetone of the equilibrium constants for the formation of pseudorotaxanes by NMR methods. The 30 new axle molecules have in common a secondary ammonium group, present as the thiocyanate salt, and an anthracen-9-ylmethyl group, but are rendered unique by the second amine substituent. All rotaxanes involve the well known polyether macrocycle, benzo[24]crown-8. The constants for the binding of axles having linear groups ranging from 2 to 18 carbon atoms show little variation in binding constant but are divided into two groups by their equilibration rates. Those with less than five methylene groups react rapidly on the NMR timescale, whereas those having more than five methylene groups are slow. Branching inhibits binding, but the effect decreases as the branch is moved away from the amine. Phenyl groups weaken binding when close to the amine but strengthen binding when more remote. Some functional groups decrease pseudorotaxane stability (alcohol functions), whereas others increase binding (carboxylic acid groups). PMID:11959934

  8. Resolving dual binding conformations of cellulosome cohesin-dockerin complexes using single-molecule force spectroscopy.

    PubMed

    Jobst, Markus A; Milles, Lukas F; Schoeler, Constantin; Ott, Wolfgang; Fried, Daniel B; Bayer, Edward A; Gaub, Hermann E; Nash, Michael A

    2015-10-31

    Receptor-ligand pairs are ordinarily thought to interact through a lock and key mechanism, where a unique molecular conformation is formed upon binding. Contrary to this paradigm, cellulosomal cohesin-dockerin (Coh-Doc) pairs are believed to interact through redundant dual binding modes consisting of two distinct conformations. Here, we combined site-directed mutagenesis and single-molecule force spectroscopy (SMFS) to study the unbinding of Coh:Doc complexes under force. We designed Doc mutations to knock out each binding mode, and compared their single-molecule unfolding patterns as they were dissociated from Coh using an atomic force microscope (AFM) cantilever. Although average bulk measurements were unable to resolve the differences in Doc binding modes due to the similarity of the interactions, with a single-molecule method we were able to discriminate the two modes based on distinct differences in their mechanical properties. We conclude that under native conditions wild-type Doc from Clostridium thermocellum exocellulase Cel48S populates both binding modes with similar probabilities. Given the vast number of Doc domains with predicted dual binding modes across multiple bacterial species, our approach opens up new possibilities for understanding assembly and catalytic properties of a broad range of multi-enzyme complexes.

  9. Single-Molecule Dynamics Reveals Cooperative Binding-Folding in Protein Recognition

    SciTech Connect

    Wang, Jin; Lu, Qiang N.; Lu, H PETER.

    2006-07-01

    The study of associations between two biomolecules is the key to understand molecular recognition and function. Molecular function is often thought to be determined by the underlying structures. Here, combining single molecule study of protein binding with an energy landscape inspired microscopic model, we found strong evidences that bio-molecular recognition is determined by flexibilities in addition to structures. Our model is based on coarse grained molecular dynamics performed on the residue level with the energy function biased towards the native binding structure (Go model). With our model, the underlying free energy landscape of the binding can be explored. Two distinct conformational states as free energy minimum, one with partially folding of CBD and significant binding of CBD to CDC42, and another with native folding of CBD and native binding of CBD to CDC42, are clearly seen. This shows the binding process proceeds with significant interface binding of CBD with CDC42 first without complete folding of CBD. Finally binding and folding are coupled with each other cooperatively to reach the native binding state. The single molecule experimental finding of the dynamic fluctuations between the loosely bound and closely bound conformational states can be identified with theoretically calculated free energy minimum and quantitatively explained in our model as a result of binding associated with large conformational changes. Theoretical predictions have identified certain key residues for binding which are consistent with mutational experiments. The combined study provides a test ground for fundamental mechanisms as well as insights into design and further explorations on biomolecular recognition with large conformational changes.

  10. A Simple Method for Improving Torsion Optimization of Ligand Molecules in Receptor Binding Sites.

    PubMed

    Che, Jianwei

    2005-07-01

    A simple but effective method is introduced for optimizing ligand molecules in torsion space within receptor binding sites. The algorithm makes use of geometric constraints of ligand molecules to search for energetically favorable conformations. It is applied to a conjugate gradient (CG) method as an example. During conformational energy optimization, new line search directions are modified according to the spatial span of rotational groups in ligand molecules. Significant improvements were observed in terms of the abilities both to recover global optimal structures and to obtain lower energy ensembles. This simple algorithm allows rapid implementation and can be incorporated into other conformational energy optimization techniques.

  11. Major histocompatibility complex class I molecules bind natural peptide ligands lacking the amino-terminal binding residue in vivo.

    PubMed

    Yague, J; Marina, A; Vazquez, J; Lopez De Castro, J A

    2001-11-23

    Major histocompatibility complex (MHC) class I-peptide complexes are stabilized by multiple interactions, including those of the peptidic NH(2)-terminal group in the A pocket of the MHC molecule. In this study, the characterization of four natural HLA-B39 ligands lacking the amino-terminal binding residue is reported. These peptides were found in the endogenous peptide pool of one or more of the B*3901, B*3905, and B*3909 allotypes and sequenced by nanoelectrospray mass spectrometry. Control experiments ruled out that they resulted from exopeptidase trimming of their NH(2)-terminally extended counterparts: NAc-SHVAVENAL, EHGPNPIL, IHEPEPHIL, and EHAGVISVL, also present in the same peptide pools, during purification. HAGVISVL and HVAVENAL behaved similarly to the corresponding NH(2)-terminally extended peptides in their binding to B*3901 and B*3909 at the cell surface in vitro, and in cell surface stabilization of B*3901. This is, to our knowledge, the first demonstration that peptides lacking the amino-terminal binding residue bind in vivo to classical MHC class I molecules. The results indicate that canonical MHC-peptide interactions in the A pocket are not always necessary for endogenous peptide presentation.

  12. Identification of small molecule binding sites within proteins using phage display technology.

    SciTech Connect

    Rodi, D. J.; Agoston, G. E.; Manon, R.; Lapcevich, R.; Green, S. J.; Makowski, L.; Biosciences Division; EntreMed Inc.; Florida State Univ.

    2001-11-01

    Affinity selection of peptides displayed on phage particles was used as the basis for mapping molecular contacts between small molecule ligands and their protein targets. Analysis of the crystal structures of complexes between proteins and small molecule ligands revealed that virtually all ligands of molecular weight 300 Da or greater have a continuous binding epitope of 5 residues or more. This observation led to the development of a technique for binding site identification which involves statistical analysis of an affinity-selected set of peptides obtained by screening of libraries of random, phage-displayed peptides against small molecules attached to solid surfaces. A random sample of the selected peptides is sequenced and used as input for a similarity scanning program which calculates cumulative similarity scores along the length of the putative receptor. Regions of the protein sequence exhibiting the highest similarity with the selected peptides proved to have a high probability of being involved in ligand binding. This technique has been employed successfully to map the contact residues in multiple known targets of the anticancer drugs paclitaxel (Taxol), docetaxel (Taxotere) and 2-methoxyestradiol and the glycosaminoglycan hyaluronan, and to identify a novel paclitaxel receptor [1]. These data corroborate the observation that the binding properties of peptides displayed on the surface of phage particles can mimic the binding properties of peptides in naturally occurring proteins. It follows directly that structural context is relatively unimportant for determining the binding properties of these disordered peptides. This technique represents a novel, rapid, high resolution method for identifying potential ligand binding sites in the absence of three-dimensional information and has the potential to greatly enhance the speed of development of novel small molecule pharmaceuticals.

  13. The Overlap of Small Molecule and Protein Binding Sites within Families of Protein Structures

    PubMed Central

    Davis, Fred P.; Sali, Andrej

    2010-01-01

    Protein–protein interactions are challenging targets for modulation by small molecules. Here, we propose an approach that harnesses the increasing structural coverage of protein complexes to identify small molecules that may target protein interactions. Specifically, we identify ligand and protein binding sites that overlap upon alignment of homologous proteins. Of the 2,619 protein structure families observed to bind proteins, 1,028 also bind small molecules (250–1000 Da), and 197 exhibit a statistically significant (p<0.01) overlap between ligand and protein binding positions. These “bi-functional positions”, which bind both ligands and proteins, are particularly enriched in tyrosine and tryptophan residues, similar to “energetic hotspots” described previously, and are significantly less conserved than mono-functional and solvent exposed positions. Homology transfer identifies ligands whose binding sites overlap at least 20% of the protein interface for 35% of domain–domain and 45% of domain–peptide mediated interactions. The analysis recovered known small-molecule modulators of protein interactions as well as predicted new interaction targets based on the sequence similarity of ligand binding sites. We illustrate the predictive utility of the method by suggesting structural mechanisms for the effects of sanglifehrin A on HIV virion production, bepridil on the cellular entry of anthrax edema factor, and fusicoccin on vertebrate developmental pathways. The results, available at http://pibase.janelia.org, represent a comprehensive collection of structurally characterized modulators of protein interactions, and suggest that homologous structures are a useful resource for the rational design of interaction modulators. PMID:20140189

  14. Binding to large enzyme pockets: small-molecule inhibitors of trypanothione reductase.

    PubMed

    Persch, Elke; Bryson, Steve; Todoroff, Nickolay K; Eberle, Christian; Thelemann, Jonas; Dirdjaja, Natalie; Kaiser, Marcel; Weber, Maria; Derbani, Hassan; Brun, Reto; Schneider, Gisbert; Pai, Emil F; Krauth-Siegel, R Luise; Diederich, François

    2014-08-01

    The causative agents of the parasitic disease human African trypanosomiasis belong to the family of trypanosomatids. These parasitic protozoa exhibit a unique thiol redox metabolism that is based on the flavoenzyme trypanothione reductase (TR). TR was identified as a potential drug target and features a large active site that allows a multitude of possible ligand orientations, which renders rational structure-based inhibitor design highly challenging. Herein we describe the synthesis, binding properties, and kinetic analysis of a new series of small-molecule inhibitors of TR. The conjunction of biological activities, mutation studies, and virtual ligand docking simulations led to the prediction of a binding mode that was confirmed by crystal structure analysis. The crystal structures revealed that the ligands bind to the hydrophobic wall of the so-called "mepacrine binding site". The binding conformation and potency of the inhibitors varied for TR from Trypanosoma brucei and T. cruzi.

  15. Synthesis of Zn-MOF incorporating titanium-hydrides as active sites binding H2 molecules

    NASA Astrophysics Data System (ADS)

    Kim, Jongsik; Ok Kim, Dong; Wook Kim, Dong; Sagong, Kil

    2015-10-01

    This paper describes the synthetic effort for a Zn-MOF imparting Ti-H as a preferential binding site potentially capturing H2 molecules via Kubas-type interaction. The formation mechanism of Ti-H innate to the final material was potentially demonstrated to follow a radical dissociation rather than a β-hydrogen elimination and a C-H reductive elimination.

  16. The use of calorimetry in the biophysical characterization of small molecule alkaloids binding to RNA structures.

    PubMed

    Kumar, Gopinatha Suresh; Basu, Anirban

    2016-05-01

    RNA has now emerged as a potential target for therapeutic intervention. RNA targeted drug design requires detailed thermodynamic characterization that provides new insights into the interactions and this together with structural data, may be used in rational drug design. The use of calorimetry to characterize small molecule-RNA interactions has emerged as a reliable and sensitive tool after the recent advancements in biocalorimetry. This review summarizes the recent advancements in thermodynamic characterization of small molecules, particularly some natural alkaloids binding to various RNA structures. Thermodynamic characterization provides information that can supplement structural data leading to more effective drug development protocols. This review provides a concise report on the use of isothermal titration calorimetry (ITC) and differential scanning calorimetry (DSC) techniques in characterizing small molecules, mostly alkaloids-RNA interactions with particular reference to binding of tRNA, single stranded RNA, double stranded RNA, poly(A), triplex RNA. It is now apparent that a combination of structural and thermodynamic data is essential for rational design of specific RNA targeted drugs. Recent advancements in biocalorimetry instrumentation have led to detailed understanding of the thermodynamics of small molecules binding to various RNA structures paving the path for the development of many new natural and synthetic molecules as specific binders to various RNA structures. RNA targeted drug design, that remained unexplored, will immensely benefit from the calorimetric studies leading to the development of effective drugs for many diseases. Copyright © 2015 Elsevier B.V. All rights reserved.

  17. Small-Molecule Modulators of Methyl-Lysine Binding for the CBX7 Chromodomain

    DOE PAGES

    Ren, Chunyan; Morohashi, Keita; Plotnikov, Alexander N.; ...

    2015-02-05

    Chromobox homolog 7 (CBX7) plays an important role in gene transcription in a wide array of cellular processes, ranging from stem cell self-renewal and differentiation to tumor progression. CBX7 functions through its N-terminal chromodomain (ChD), which recognizes tri-methylated lysine 27 of histone 3 (H3K27me3), a conserved epigenetic mark that signifies gene transcriptional repression. Here in this study, we report discovery of small molecules that inhibit CBX7ChD binding to H3K27me3. Our crystal structures reveal the binding modes of these molecules that compete against H3K27me3 binding through interactions with key residues in the methyl-lysine binding pocket of CBX7ChD. We further show thatmore » a lead compound MS37452, derepresses transcription of Polycomb repressive complex target gene p16/CDKN2A by displacing CBX7 binding to the INK4A/ARF locus in prostate cancer cells. Ultimately, these small molecules have the potential to be developed into high-potency chemical modulators that target CBX7 functions in gene transcription in different disease pathways.« less

  18. Small-molecule modulators of methyl-lysine binding for the CBX7 chromodomain.

    PubMed

    Ren, Chunyan; Morohashi, Keita; Plotnikov, Alexander N; Jakoncic, Jean; Smith, Steven G; Li, Jiaojie; Zeng, Lei; Rodriguez, Yoel; Stojanoff, Vivian; Walsh, Martin; Zhou, Ming-Ming

    2015-02-19

    Chromobox homolog 7 (CBX7) plays an important role in gene transcription in a wide array of cellular processes, ranging from stem cell self-renewal and differentiation to tumor progression. CBX7 functions through its N-terminal chromodomain (ChD), which recognizes trimethylated lysine 27 of histone 3 (H3K27me3), a conserved epigenetic mark that signifies gene transcriptional repression. In this study, we report the discovery of small molecules that inhibit CBX7ChD binding to H3K27me3. Our crystal structures reveal the binding modes of these molecules that compete against H3K27me3 binding through interactions with key residues in the methyl-lysine binding pocket of CBX7ChD. We further show that a lead compound, MS37452, derepresses transcription of Polycomb repressive complex target gene p16/CDKN2A by displacing CBX7 binding to the INK4A/ARF locus in prostate cancer cells. These small molecules have the potential to be developed into high-potency chemical modulators that target CBX7 functions in gene transcription in different disease pathways. Copyright © 2015 Elsevier Ltd. All rights reserved.

  19. Binding modes of thioflavin T molecules to prion peptide assemblies identified by using scanning tunneling microscopy.

    PubMed

    Mao, Xiaobo; Guo, Yuanyuan; Wang, Chenxuan; Zhang, Min; Ma, Xiaojing; Liu, Lei; Niu, Lin; Zeng, Qingdao; Yang, Yanlian; Wang, Chen

    2011-06-15

    The widely used method to monitor the aggregation process of amyloid peptide is thioflavin T (ThT) assay, while the detailed molecular mechanism is still not clear. In this work, we report here the direct identification of the binding modes of ThT molecules with the prion peptide GNNQQNY by using scanning tunneling microscopy (STM). The assembly structures of GNNQQNY were first observed by STM on a graphite surface, and the introduction of ThT molecules to the surface facilitated the STM observations of the adsorption conformations of ThT with peptide strands. ThT molecules are apt to adsorb on the peptide assembly with β-sheet structure and oriented parallel with the peptide strands adopting four different binding modes. This effort could benefit the understanding of the mechanisms of the interactions between labeling species or inhibitory ligands and amyloid peptides, which is keenly needed for developing diagnostic and therapeutic approaches.

  20. Natural alkaloid Luotonin A and its affixed acceptor molecules: Serum albumin binding studies.

    PubMed

    Kesavan, Mookkandi Palsamy; Kumar, Gujuluva Gangatharan Vinoth; Anitha, Kandasamy; Ravi, Lokesh; Raja, Jeyaraj Dhaveethu; Rajagopal, Gurusamy; Rajesh, Jegathalaprathaban

    2017-08-01

    Effective interaction of natural alkaloid Luotonin A (L) and its affixed acceptor molecules 1 and 2 with donor molecule as Bovine serum albumin (BSA) at various pH (4.0, 7.4 and 10.0) medium have been demonstrated using various conventional spectroscopic techniques. These analyses provide some valuable features on the interaction between BSA and acceptor molecules (L, 1 and 2). From the absorption and fluorescence spectral titration studies, the formation of ground-state complexes between the acceptor molecules (L, 1 and 2) and the BSA have been confirmed. The results of the afore titrations analysis reveal that, the strong binding of receptor 1 with BSA (Kapp 5.68×10(4)M(-1); KSV 1.86×10(6)Lmol(-1); Ka 6.42×10(5)Lmol(-1); Kass 8.09×10(6)M(-1); ΔG -33.35kJ/mol) at physiological pH medium (7.4) than other receptor molecules 2 and L. The Förster resonance energy transfer (FRET) efficiency between the tryptophan (Trp) residues of BSA and acceptor molecules L, 1 and 2 during the interaction, are 28.85, 85.24 and 53.25 % respectively. The superior binding efficacy of acceptor 1 at physiological pH condition has been further confirmed by FT-IR and Raman spectral analysis methods. Moreover, theoretical docking studies of acceptors L, 1 and 2 towards HSA have been demonstrated to differentiate their binding behaviours. It reveals that, acceptor 1 has the strongest binding ability with HSA through two hydrogen bonding and the Atomic contact energy (ACE) value of -483.96kcal/mol. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. DEPTH: a web server to compute depth and predict small-molecule binding cavities in proteins.

    PubMed

    Tan, Kuan Pern; Varadarajan, Raghavan; Madhusudhan, M S

    2011-07-01

    Depth measures the extent of atom/residue burial within a protein. It correlates with properties such as protein stability, hydrogen exchange rate, protein-protein interaction hot spots, post-translational modification sites and sequence variability. Our server, DEPTH, accurately computes depth and solvent-accessible surface area (SASA) values. We show that depth can be used to predict small molecule ligand binding cavities in proteins. Often, some of the residues lining a ligand binding cavity are both deep and solvent exposed. Using the depth-SASA pair values for a residue, its likelihood to form part of a small molecule binding cavity is estimated. The parameters of the method were calibrated over a training set of 900 high-resolution X-ray crystal structures of single-domain proteins bound to small molecules (molecular weight <1.5  KDa). The prediction accuracy of DEPTH is comparable to that of other geometry-based prediction methods including LIGSITE, SURFNET and Pocket-Finder (all with Matthew's correlation coefficient of ∼0.4) over a testing set of 225 single and multi-chain protein structures. Users have the option of tuning several parameters to detect cavities of different sizes, for example, geometrically flat binding sites. The input to the server is a protein 3D structure in PDB format. The users have the option of tuning the values of four parameters associated with the computation of residue depth and the prediction of binding cavities. The computed depths, SASA and binding cavity predictions are displayed in 2D plots and mapped onto 3D representations of the protein structure using Jmol. Links are provided to download the outputs. Our server is useful for all structural analysis based on residue depth and SASA, such as guiding site-directed mutagenesis experiments and small molecule docking exercises, in the context of protein functional annotation and drug discovery.

  2. DEPTH: a web server to compute depth and predict small-molecule binding cavities in proteins

    PubMed Central

    Tan, Kuan Pern; Varadarajan, Raghavan; Madhusudhan, M. S.

    2011-01-01

    Depth measures the extent of atom/residue burial within a protein. It correlates with properties such as protein stability, hydrogen exchange rate, protein–protein interaction hot spots, post-translational modification sites and sequence variability. Our server, DEPTH, accurately computes depth and solvent-accessible surface area (SASA) values. We show that depth can be used to predict small molecule ligand binding cavities in proteins. Often, some of the residues lining a ligand binding cavity are both deep and solvent exposed. Using the depth-SASA pair values for a residue, its likelihood to form part of a small molecule binding cavity is estimated. The parameters of the method were calibrated over a training set of 900 high-resolution X-ray crystal structures of single-domain proteins bound to small molecules (molecular weight <1.5 KDa). The prediction accuracy of DEPTH is comparable to that of other geometry-based prediction methods including LIGSITE, SURFNET and Pocket-Finder (all with Matthew’s correlation coefficient of ∼0.4) over a testing set of 225 single and multi-chain protein structures. Users have the option of tuning several parameters to detect cavities of different sizes, for example, geometrically flat binding sites. The input to the server is a protein 3D structure in PDB format. The users have the option of tuning the values of four parameters associated with the computation of residue depth and the prediction of binding cavities. The computed depths, SASA and binding cavity predictions are displayed in 2D plots and mapped onto 3D representations of the protein structure using Jmol. Links are provided to download the outputs. Our server is useful for all structural analysis based on residue depth and SASA, such as guiding site-directed mutagenesis experiments and small molecule docking exercises, in the context of protein functional annotation and drug discovery. PMID:21576233

  3. Photochemically enhanced binding of small molecules to the tumor necrosis factor receptor-1 inhibits the binding of TNF-[alpha

    SciTech Connect

    Carter, Percy H.; Scherle, Peggy A.; Muckelbauer, Jodi K.; Voss, Matthew E.; Liu, Rui-qin; Thompson III, Lorin A.; Xu, Meizhong; Lo, Yvonne C.; Li, Zhong; Strzemienski, Paul; Yang, Gengjie; Falahatpishen, Nikoo; Farrow, Neil A.; Tebben, Andrew J.; Underwood, Denis; Trzaskos, James M.; Friedman, Steven M.; Newton, Robert C.; Decicco, Carl P.

    2010-03-05

    The binding of tumor necrosis factor alpha (TNF-{alpha}) to the type-1 TNF receptor (TNFRc1) plays an important role in inflammation. Despite the clinical success of biologics (antibodies, soluble receptors) for treating TNF-based autoimmune conditions, no potent small molecule antagonists have been developed. Our screening of chemical libraries revealed that N-alkyl 5-arylidene-2-thioxo-1,3-thiazolidin-4-ones were antagonists of this protein-protein interaction. After chemical optimization, we discovered IW927, which potently disrupted the binding of TNF-{alpha} to TNFRc1 (IC{sub 50} = 50 nM) and also blocked TNF-stimulated phosphorylation of I{kappa}-B in Ramos cells (IC{sub 50} = 600 nM). This compound did not bind detectably to the related cytokine receptors TNFRc2 or CD40, and did not display any cytotoxicity at concentrations as high as 100 {micro}M. Detailed evaluation of this and related molecules revealed that compounds in this class are 'photochemically enhanced' inhibitors, in that they bind reversibly to the TNFRc1 with weak affinity (ca. 40-100 mM) and then covalently modify the receptor via a photochemical reaction. We obtained a crystal structure of IV703 (a close analog of IW927) bound to the TNFRc1. This structure clearly revealed that one of the aromatic rings of the inhibitor was covalently linked to the receptor through the main-chain nitrogen of Ala-62, a residue that has already been implicated in the binding of TNF-{alpha} to the TNFRc1. When combined with the fact that our inhibitors are reversible binders in light-excluded conditions, the results of the crystallography provide the basis for the rational design of nonphotoreactive inhibitors of the TNF-{alpha}-TNFRc1 interaction.

  4. Photochemically enhanced binding of small molecules to the tumor necrosis factor receptor-1 inhibits the binding of TNF-alpha.

    PubMed

    Carter, P H; Scherle, P A; Muckelbauer, J K; Voss, M E; Liu, R Q; Thompson, L A; Tebben, A J; Solomon, K A; Lo, Y C; Li, Z; Strzemienski, P; Yang, G; Falahatpisheh, N; Xu, M; Wu, Z; Farrow, N A; Ramnarayan, K; Wang, J; Rideout, D; Yalamoori, V; Domaille, P; Underwood, D J; Trzaskos, J M; Friedman, S M; Newton, R C; Decicco, C P; Muckelbauer, J A

    2001-10-09

    The binding of tumor necrosis factor alpha (TNF-alpha) to the type-1 TNF receptor (TNFRc1) plays an important role in inflammation. Despite the clinical success of biologics (antibodies, soluble receptors) for treating TNF-based autoimmune conditions, no potent small molecule antagonists have been developed. Our screening of chemical libraries revealed that N-alkyl 5-arylidene-2-thioxo-1,3-thiazolidin-4-ones were antagonists of this protein-protein interaction. After chemical optimization, we discovered IW927, which potently disrupted the binding of TNF-alpha to TNFRc1 (IC(50) = 50 nM) and also blocked TNF-stimulated phosphorylation of Ikappa-B in Ramos cells (IC(50) = 600 nM). This compound did not bind detectably to the related cytokine receptors TNFRc2 or CD40, and did not display any cytotoxicity at concentrations as high as 100 microM. Detailed evaluation of this and related molecules revealed that compounds in this class are "photochemically enhanced" inhibitors, in that they bind reversibly to the TNFRc1 with weak affinity (ca. 40-100 microM) and then covalently modify the receptor via a photochemical reaction. We obtained a crystal structure of IV703 (a close analog of IW927) bound to the TNFRc1. This structure clearly revealed that one of the aromatic rings of the inhibitor was covalently linked to the receptor through the main-chain nitrogen of Ala-62, a residue that has already been implicated in the binding of TNF-alpha to the TNFRc1. When combined with the fact that our inhibitors are reversible binders in light-excluded conditions, the results of the crystallography provide the basis for the rational design of nonphotoreactive inhibitors of the TNF-alpha-TNFRc1 interaction.

  5. Toxic shock syndrome toxin 1 binds to major histocompatibility complex class II molecules.

    PubMed Central

    Scholl, P; Diez, A; Mourad, W; Parsonnet, J; Geha, R S; Chatila, T

    1989-01-01

    Toxic shock syndrome toxin 1 (TSST-1) is a 22-kDa exotoxin produced by strains of Staphylococcus aureus and implicated in the pathogenesis of toxic shock syndrome. In common with other staphylococcal exotoxins, TSST-1 has diverse immunological effects. These include the induction of interleukin 2 receptor expression, interleukin 2 synthesis, proliferation of human T lymphocytes, and stimulation of interleukin 1 synthesis by human monocytes. In the present study, we demonstrate that TSST-1 binds with saturation kinetics and with a dissociation constant of 17-43 nM to a single class of binding sites on human mononuclear cells. There was a strong correlation between the number of TSST-1 binding sites and the expression of major histocompatibility complex class II molecules, and interferon-gamma induced the expression of class II molecules as well as TSST-1 binding sites on human skin-derived fibroblasts. Monoclonal antibodies to HLA-DR, but not to HLA-DP or HLA-DQ, strongly inhibited TSST-1 binding. Affinity chromatography of 125I-labeled cell membranes over TSST-1-agarose resulted in the recovery of two bands of 35 kDa and 31 kDa that comigrated, respectively, with the alpha and beta chains of HLA-DR and that could be immunoprecipitated with anti-HLA-DR monoclonal antibodies. Binding of TSST-1 was demonstrated to HLA-DR and HLA-DQ L-cell transfectants. These results indicate that major histocompatibility complex class II molecules represent the major binding site for TSST-1 on human cells. Images PMID:2542966

  6. A single-molecule approach to explore binding, uptake and transport of cancer cell targeting nanotubes

    NASA Astrophysics Data System (ADS)

    Lamprecht, C.; Plochberger, B.; Ruprecht, V.; Wieser, S.; Rankl, C.; Heister, E.; Unterauer, B.; Brameshuber, M.; Danzberger, J.; Lukanov, P.; Flahaut, E.; Schütz, G.; Hinterdorfer, P.; Ebner, A.

    2014-03-01

    In the past decade carbon nanotubes (CNTs) have been widely studied as a potential drug-delivery system, especially with functionality for cellular targeting. Yet, little is known about the actual process of docking to cell receptors and transport dynamics after internalization. Here we performed single-particle studies of folic acid (FA) mediated CNT binding to human carcinoma cells and their transport inside the cytosol. In particular, we employed molecular recognition force spectroscopy, an atomic force microscopy based method, to visualize and quantify docking of FA functionalized CNTs to FA binding receptors in terms of binding probability and binding force. We then traced individual fluorescently labeled, FA functionalized CNTs after specific uptake, and created a dynamic ‘roadmap’ that clearly showed trajectories of directed diffusion and areas of nanotube confinement in the cytosol. Our results demonstrate the potential of a single-molecule approach for investigation of drug-delivery vehicles and their targeting capacity.

  7. Distribution of binding energies of a water molecule in the water liquid-vapor interface

    SciTech Connect

    Chempath, Shaji; Pratt, Lawrence R

    2008-01-01

    Distributions of binding energies of a water molecule in the water liquid-vapor interface are obtained on the basis of molecular simulation with the SPC/E model of water. These binding energies together with the observed interfacial density profile are used to test a minimally conditioned Gaussian quasi-chemical statistical thermodynamic theory. Binding energy distributions for water molecules in that interfacial region clearly exhibit a composite structure. A minimally conditioned Gaussian quasi-chemical model that is accurate for the free energy of bulk liquid water breaks down for water molecules in the liquid-vapor interfacial region. This breakdown is associated with the fact that this minimally conditioned Gaussian model would be inaccurate for the statistical thermodynamics of a dilute gas. Aggressive conditioning greatly improves the performance of that Gaussian quasi-chemical model. The analogy between the Gaussian quasi-chemical model and dielectric models of hydration free energies suggests that naive dielectric models without the conditioning features of quasi-chemical theory will be unreliable for these interfacial problems. Multi-Gaussian models that address the composite nature of the binding energy distributions observed in the interfacial region might provide a mechanism for correcting dielectric models for practical applications.

  8. Quantum dot binding to DNA: single-molecule imaging with atomic force microscopy.

    PubMed

    Li, Kungang; Zhang, Wen; Chen, Yongsheng

    2013-01-01

    The interaction between nanoparticles (NPs) and DNA is of significance for both application and implication research of NPs. In this study, a single-molecule imaging technique based on atomic force microscopy (AFM) was employed to probe the NP-DNA interactions with quantum dots (QDs) as model NPs. Reproducible high-quality images of single DNA molecules in air and in liquids were acquired on mica by optimizing sample preparation conditions. Furthermore, the binding of QDs to DNA was explored using AFM. The DNA concentration was found to be a key factor influencing AFM imaging quality. In air and liquids, the optimal DNA concentration for imaging DNA molecules was approximately 2.5 and 0.25 μg/mL, and that for imaging DNA binding with QDs was 0.5 and 0.25 μg/mL, respectively. In the presence of QDs, the DNA conformation was altered with the formation of DNA condensates. Finally, the fine conformation of QD-DNA binding sites was examined to analyze the binding mechanisms. This work will benefit investigations of NP-DNA interactions and the understanding of the structure of NP-DNA bioconjugates. See accompanying article by Wang DOI: 10.1002/biot.201200309. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Identification of C3b-Binding Small-Molecule Complement Inhibitors Using Cheminformatics.

    PubMed

    Garcia, Brandon L; Skaff, D Andrew; Chatterjee, Arindam; Hanning, Anders; Walker, John K; Wyckoff, Gerald J; Geisbrecht, Brian V

    2017-05-01

    The complement system is an elegantly regulated biochemical cascade formed by the collective molecular recognition properties and proteolytic activities of more than two dozen membrane-bound or serum proteins. Complement plays diverse roles in human physiology, such as acting as a sentry against invading microorganisms, priming of the adaptive immune response, and removal of immune complexes. However, dysregulation of complement can serve as a trigger for a wide range of human diseases, which include autoimmune, inflammatory, and degenerative conditions. Despite several potential advantages of modulating complement with small-molecule inhibitors, small-molecule drugs are highly underrepresented in the current complement-directed therapeutics pipeline. In this study, we have employed a cheminformatics drug discovery approach based on the extensive structural and functional knowledge available for the central proteolytic fragment of the cascade, C3b. Using parallel in silico screening methodologies, we identified 45 small molecules that putatively bind C3b near ligand-guided functional hot spots. Surface plasmon resonance experiments resulted in the validation of seven dose-dependent C3b-binding compounds. Competition-based biochemical assays demonstrated the ability of several C3b-binding compounds to interfere with binding of the original C3b ligand that guided their discovery. In vitro assays of complement function identified a single complement inhibitory compound, termed cmp-5, and mechanistic studies of the cmp-5 inhibitory mode revealed it acts at the level of C5 activation. This study has led to the identification of a promising new class of C3b-binding small-molecule complement inhibitors and, to our knowledge, provides the first demonstration of cheminformatics-based, complement-directed drug discovery. Copyright © 2017 by The American Association of Immunologists, Inc.

  10. Label-free detection of small-molecule binding to a GPCR in the membrane environment.

    PubMed

    Heym, Roland G; Hornberger, Wilfried B; Lakics, Viktor; Terstappen, Georg C

    2015-08-01

    Evaluation of drug-target interaction kinetics is becoming increasingly important during the drug-discovery process to investigate selectivity of a drug and predict in vivo target occupancy. To date, it remains challenging to obtain kinetic information for interactions between G-protein-coupled receptors (GPCRs) and small-molecule ligands in a label-free manner. Often GPCRs need to be solubilized or even stabilized by mutations which can be difficult and is time consuming. In addition, it is often unclear if the native conformation of the receptors is sustained. In this study, surface plasmon resonance (SPR) and surface acoustic wave (SAW) technologies have been used to detect ligand binding to the GPCR chemokine (C-X-C motif) receptor 4 (CXCR4) expressed in lipoparticles. We first evaluated different strategies to immobilize CXCR4-expressing lipoparticles. The highest small-molecule binding signal in SPR and SAW was achieved with a matrix-free carboxymethylated sensor chip coated with wheat germ agglutinin for lipoparticle capturing. Next, the binding kinetics of the anti-CXCR4 antibody 12G5 raised against a conformational epitope (k(on)=1.83×10(6)M(-1)s(-1), k(off)=2.79×10(-4) s(-1)) and the small molecule AMD3100 (k(on)=5.46×10(5)M(-1)s(-1), k(off)=1.01×10(-2)s(-1)) were assessed by SAW. Our kinetic and affinity data are consistent with previously published radioligand-binding experiments using cells and label-free experiments with solubilized CXCR4. This is the first study demonstrating label-free kinetic characterization of small-molecule binding to a GPCR in the membrane environment. The presented method holds the potential to greatly facilitate label-free assay development for GPRCs that can be expressed at high levels in lipoparticles.

  11. Preferable binding site of gas molecules on graphene nanoribbon with Stone–Wales defect

    NASA Astrophysics Data System (ADS)

    Auzar, Zuriana; Johari, Zaharah; Sakina, S. H.; Alias, N. E.; Abidin, M. S. Z.

    2017-02-01

    The issue of sensitivity of sensing device has focused on the development of sensing devices by using new materials, such as graphene. The gas molecules in different positions such as on, near and far from the defect are placed in the same binding site in two graphene configurations for fair comparison. The interaction between two different graphene configurations such as (pristine armchair graphene nanoribbon (P-AGNR) and Stone–Wales defect on graphene surface (SW-AGNR)) with gas molecules (e.g. O2, N2 and NH3) have been investigated to observe the preferential position site of adsorbate gas molecules. The preferable position sites are investigated by using Extended-Huckel Theory. It is found that, the electronic properties of each configuration are strongly depends on the position of gas molecules and graphene system. Meanwhile, the binding site of the gas molecules on the defective site of graphene surface is a significant factor in determining the sensing behavior of graphene based gas defection device.

  12. Subtle conformational changes induced in major histocompatibility complex class II molecules by binding peptides.

    PubMed

    Chervonsky, A V; Medzhitov, R M; Denzin, L K; Barlow, A K; Rudensky, A Y; Janeway, C A

    1998-08-18

    Intracellular trafficking of major histocompatibility complex (MHC) class II molecules is characterized by passage through specialized endocytic compartment(s) where antigenic peptides replace invariant chain fragments in the presence of the DM protein. These changes are accompanied by structural transitions of the MHC molecules that can be visualized by formation of compact SDS-resistant dimers, by changes in binding of mAbs, and by changes in T cell responses. We have observed that a mAb (25-9-17) that is capable of staining I-Ab on the surface of normal B cells failed to interact with I-Ab complexes with a peptide derived from the Ealpha chain of the I-E molecule but bound a similar covalent complex of I-Ab with the class II binding fragment (class II-associated invariant chain peptides) of the invariant chain. Moreover, 25-9-17 blocked activation of several I-Ab-reactive T cell hybridomas but failed to block others, suggesting that numerous I-Ab-peptide complexes acquire the 25-9-17(+) or 25-9-17(-) conformation. Alloreactive T cells were also able to discriminate peptide-dependent variants of MHC class II molecules. Thus, peptides impose subtle structural transitions upon MHC class II molecules that affect T cell recognition and may thus be critical for T cell selection and autiommunity.

  13. First-principles Hubbard U approach for small molecule binding in metal-organic frameworks

    NASA Astrophysics Data System (ADS)

    Mann, Gregory W.; Lee, Kyuho; Cococcioni, Matteo; Smit, Berend; Neaton, Jeffrey B.

    2016-05-01

    We apply first-principles approaches with Hubbard U corrections for calculation of small molecule binding energetics to open-shell transition metal atoms in metal-organic frameworks (MOFs). Using density functional theory with van der Waals dispersion-corrected functionals, we determine Hubbard U values ab initio through an established linear response procedure for M-MOF-74, for a number of different metal centers (M = Ti, V, Cr, Mn, Fe, Co, Ni, and Cu). While our ab initio U values differ from those used in previous work, we show that they result in lattice parameters and electronic contributions to CO2-MOF binding energies that lead to excellent agreement with experiments and previous results, yielding lattice parameters within 3%. In addition, U-dependent calculations for an example system, Co-MOF-74, suggest that the CO2 binding energy grows monotonically with the value of Hubbard U, with the binding energy shifting 4 kJ/mol (or 0.041 eV) over the range of U = 0-5.4 eV. These results provide insight into an approximate but computationally efficient means for calculation of small molecule binding energies to open-shell transition metal atoms in MOFs and suggest that the approach can be predictive with good accuracy, independent of the cations used and the availability of experimental data.

  14. Analysis of Small Molecule Ligands Targeting the HIV-1 Matrix Protein-RNA Binding Site*

    PubMed Central

    Alfadhli, Ayna; McNett, Henry; Eccles, Jacob; Tsagli, Seyram; Noviello, Colleen; Sloan, Rachel; López, Claudia S.; Peyton, David H.; Barklis, Eric

    2013-01-01

    The matrix domain (MA) of the HIV-1 precursor Gag (PrGag) protein directs PrGag proteins to assembly sites at the plasma membrane by virtue of its affinity to the phospholipid, phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2). MA also binds to RNA at a site that overlaps its PI(4,5)P2 site, suggesting that RNA binding may protect MA from associating with inappropriate cellular membranes prior to PrGag delivery to the PM. Based on this, we have developed an assay in which small molecule competitors to MA-RNA binding can be characterized, with the assumption that such compounds might interfere with essential MA functions and help elucidate additional features of MA binding. Following this approach, we have identified four compounds, including three thiadiazolanes, that compete with RNA for MA binding. We also have identified MA residues involved in thiadiazolane binding and found that they overlap the MA PI(4,5)P2 and RNA sites. Cell culture studies demonstrated that thiadiazolanes inhibit HIV-1 replication but are associated with significant levels of toxicity. Nevertheless, these observations provide new insights into MA binding and pave the way for the development of antivirals that target the HIV-1 matrix domain. PMID:23135280

  15. Effects of water molecules on binding kinetics of peptide receptor on a piezoelectric microcantilever

    NASA Astrophysics Data System (ADS)

    Hui Kim, Sang; Kyoung Yoo, Yong; Chae, Myung-Sic; Yoon Kang, Ji; Song Kim, Tae; Seon Hwang, Kyo; Hoon Lee, Jeong

    2012-12-01

    The use of highly selective reversible peptide receptors is essential for cantilever-based electronic nose systems. Here, we present the effects of water molecules on the binding kinetics of 2,4-dinitrotoluene (DNT) molecules with DNT selective peptide receptors linked with a tri(ethylene glycol)-based (TEG) self-assembled monolayer (SAM) in a gas phase in a piezoelectric microcantilever sensor. We observed 1.5-times faster reaction kinetics in wet conditions compared with dry conditions. In a dissociation step, distinctive differences in the recovery time were observed in wet conditions, which could be attributed to water retention efficiency of TEG-linkers for the conformation of biomolecules.

  16. Apparent lack of MHC restriction in binding of class I HLA molecules to solid-phase peptides

    PubMed Central

    1990-01-01

    The specificity of binding of solubilized, purified HLA-A,B molecules to solid-phase peptides has been examined using the assay described by Bouillet et al. [1989. Nature (Lond.). 339:473.] 64 peptides derived from the sequences of viral antigens, HLA-A,B,C heavy chains, and clathrin light chains were tested for binding to HLA-A2.1, Aw68.1, Aw69, B44, and B5, molecules that differ by 5-17 residues of the peptide binding groove. 15 of the peptides, including those known to be T cell epitopes, gave significant binding. The pattern of peptide binding for each of the five HLA-A,B molecules was not significantly different. Binding was demonstrated to be a property of native beta 2m- associated HLA-A,B molecules that preserved conformational antigenic determinants after binding to peptide. In comparison to our previous results from solution-based assays the proportion of HLA-A,B molecules that can bind solid-phase peptides is very high. This accessibility of solid-phase peptides to the binding site of MHC molecules may be directly related to the observed absence of MHC specificity in the binding. PMID:1696957

  17. Direct binding of a redox protein for single-molecule electron transfer measurements.

    PubMed

    Della Pia, Eduardo A; Macdonald, J Emyr; Elliott, Martin; Jones, D Dafydd

    2012-08-06

    An electron transfer protein is engineered with two thiol groups introduced at different positions in the molecular structure to allow robust binding to two gold electrodes. Atomic force microscopy and scanning tunneling microscopy single-molecule studies show that the engineered proteins: (1) bind to a gold electrode in defined orientation dictated by the thiol-pair utilised, and (2) have a higher conductance than the wild-type proteins indicating a more efficient electron transmission due to the strong gold-thiol contacts. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Applications of Engineered DNA-Binding Molecules Such as TAL Proteins and the CRISPR/Cas System in Biology Research.

    PubMed

    Fujita, Toshitsugu; Fujii, Hodaka

    2015-09-24

    Engineered DNA-binding molecules such as transcription activator-like effector (TAL or TALE) proteins and the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) (CRISPR/Cas) system have been used extensively for genome editing in cells of various types and species. The sequence-specific DNA-binding activities of these engineered DNA-binding molecules can also be utilized for other purposes, such as transcriptional activation, transcriptional repression, chromatin modification, visualization of genomic regions, and isolation of chromatin in a locus-specific manner. In this review, we describe applications of these engineered DNA-binding molecules for biological purposes other than genome editing.

  19. Computational study of small molecule binding for both tethered and free conditions

    PubMed Central

    2010-01-01

    Using a calix[4]arene-benzene complex as a test system we compare the potential of mean force for when the calix[4]arene is tethered versus free. When the complex is in vacuum our results show that the difference between tethered and free is primarily due to the entropic contribution to the potential of mean force resulting in a significant binding free energy difference of 6.6 kJ/mol. By contrast, when the complex is in water our results suggest that there is no appreciable difference between tethered and free. This study elucidates the roles of entropy and enthalpy for this small molecule system and emphasizes the point that tethering the receptor has the potential to dramatically impact the binding properties. These findings should be taken into consideration when using calixarene molecules in nanosensor design. PMID:20369865

  20. The role of anchor residues in the binding of peptides to HLA-A*1101 molecules.

    PubMed

    Chujoh, Y; Sobao, Y; Miwa, K; Kaneko, Y; Takiguchi, M

    1998-12-01

    The binding of 136 8- to 12-mer peptides carrying anchor residues at position 2 (P2) and the C-terminus to HLA-A*1101 molecules was analyzed by a stabilization assay using RMA-S transfectants expressing HLA-A*1101 and human beta2-microglobulin. 72.1% of these peptides bound to HLA-A*1101 molecules. Two known HLA-All-restricted cytotoxic T-lymphocyte epitope peptides showed high affinity to HLA-A*1101. The results confirmed a previous pool sequencing study of HLA-A*1101 binding self-peptides, which showed that Lys at the C-terminus and Val, Ile, Phe, Tyr, and Thr at P2 are anchor residues for HLA-A*1101. Thr and aliphatic hydrophobic residues Val, Ile, and Leu at P2 are stronger anchor residues than the aromatic hydrophobic residues Phe and Tyr. In addition, hydrophobic residues Leu, Phe, Tyr, Ile, and Ala at position 3 (P3) are secondary anchors but are weaker than those at P2. The affinities of the 8- and 12-mer peptides were significantly lower than those of 9- to 11-mer peptides. There was however no difference in affinity between 9-, 10- and 11-mer peptides. Furthermore, the analysis using peptides mutated at the C-terminus showed that HLA-A*1101 molecules can bind peptides carrying another positively charged residue, Arg. The present study clarified the role of the anchor residues at P2, P3 and the C-terminus in the binding of HLA-A*1101 molecules.

  1. Distinct effect of xenobiotics on the metal-binding properties of protein molecules.

    PubMed

    Novikova, Natalia; Kovalchuk, Mikhail; Stepina, Nina; Gaynutdinov, Radmir; Chukhrai, Elena; Yurieva, Eleonora

    2015-07-01

    The X-ray standing-wave method was applied to study the elemental composition and molecular organization of ordered protein films of alkaline phosphatase exposed to different xenobiotics (drug compounds, lead). Binding of metal ions from triply distilled water to protein molecules has been experimentally observed. Definite differences in the arrangement of impurity metal ions in the films have been established. The considerable enhancement of protein-metal interactions is attributed to partial rearrangement of the protein native structure, induced by xenobiotics.

  2. Both host and parasite MIF molecules bind to chicken macrophages via CD74 surface receptor.

    PubMed

    Kim, Sungwon; Cox, Chasity M; Jenkins, Mark C; Fetterer, Ray H; Miska, Katarzyna B; Dalloul, Rami A

    2014-12-01

    Macrophage migration inhibitory factor (MIF) is recognized as a soluble protein that inhibits the random migration of macrophages and plays a pivotal immunoregulatory function in innate and adaptive immunity. Our group has identified both chicken and Eimeria MIFs, and characterized their function in enhancing innate immune responses during inflammation. In this study, we report that chicken CD74 (ChCD74), a type II transmembrane protein, functions as a macrophage surface receptor that binds to MIF molecules. First, to examine the binding of MIF to chicken monocytes/macrophages, fresh isolated chicken peripheral blood mononuclear cells (PBMCs) were stimulated with rChIFN-γ and then incubated with recombinant chicken MIF (rChMIF). Immunofluorescence staining with anti-ChMIF followed by flow cytometry revealed the binding of MIF to stimulated PBMCs. To verify that ChCD74 acts as a surface receptor for MIF molecules, full-length ChCD74p41 was cloned, expressed and its recombinant protein (rChCD74p41) transiently over-expressed with green fluorescent protein in chicken fibroblast DF-1 cells. Fluorescence analysis revealed a higher population of cells double positive for CD74p41 and rChMIF, indicating the binding of rChMIF to DF-1 cells via rChCD74p41. Using a similar approach, it was found that Eimeria MIF (EMIF), which is secreted by Eimeria sp. during infection, bound to chicken macrophages via ChCD74p41 as a surface receptor. Together, this study provides conclusive evidence that both host and parasite MIF molecules bind to chicken macrophages via the surface receptor ChCD74.

  3. Single-Molecule Counting of Point Mutations by Transient DNA Binding

    PubMed Central

    Su, Xin; Li, Lidan; Wang, Shanshan; Hao, Dandan; Wang, Lei; Yu, Changyuan

    2017-01-01

    High-confidence detection of point mutations is important for disease diagnosis and clinical practice. Hybridization probes are extensively used, but are hindered by their poor single-nucleotide selectivity. Shortening the length of DNA hybridization probes weakens the stability of the probe-target duplex, leading to transient binding between complementary sequences. The kinetics of probe-target binding events are highly dependent on the number of complementary base pairs. Here, we present a single-molecule assay for point mutation detection based on transient DNA binding and use of total internal reflection fluorescence microscopy. Statistical analysis of single-molecule kinetics enabled us to effectively discriminate between wild type DNA sequences and single-nucleotide variants at the single-molecule level. A higher single-nucleotide discrimination is achieved than in our previous work by optimizing the assay conditions, which is guided by statistical modeling of kinetics with a gamma distribution. The KRAS c.34 A mutation can be clearly differentiated from the wild type sequence (KRAS c.34 G) at a relative abundance as low as 0.01% mutant to WT. To demonstrate the feasibility of this method for analysis of clinically relevant biological samples, we used this technology to detect mutations in single-stranded DNA generated from asymmetric RT-PCR of mRNA from two cancer cell lines. PMID:28262827

  4. Binding and diffusion of CheR molecules within a cluster of membrane receptors.

    PubMed

    Levin, Matthew D; Shimizu, Thomas S; Bray, Dennis

    2002-04-01

    Adaptation of the attractant response in Escherichia coli is attributable to the methylation of its transmembrane chemotactic receptors by the methyltransferase CheR. This protein contains two binding domains, one for the sites of methylation themselves and the other for a flexible tether at the C terminus of the receptor. We have explored the theoretical consequences of this binding geometry for a CheR molecule associated with a cluster of chemotactic receptors. Calculations show that the CheR molecule will bind with high net affinity to the receptor lattice, having a high probability of being attached by one or both of its domains at any instant of time. Because of the relatively low affinity of its individual domains and the close proximity of neighboring receptors, it is likely that when one domain unbinds it will reattach to the array before the other domain unbinds. Stochastic simulations show that the enzyme will move through the receptor cluster in a hand-over-hand fashion, like a gibbon swinging through the branches of a tree. We explore the possible consequences of this motion, which we term "molecular brachiation", for chemotactic adaptation and suggest that a similar mechanism may be operative in other large assemblies of protein molecules.

  5. Binding and diffusion of CheR molecules within a cluster of membrane receptors.

    PubMed Central

    Levin, Matthew D; Shimizu, Thomas S; Bray, Dennis

    2002-01-01

    Adaptation of the attractant response in Escherichia coli is attributable to the methylation of its transmembrane chemotactic receptors by the methyltransferase CheR. This protein contains two binding domains, one for the sites of methylation themselves and the other for a flexible tether at the C terminus of the receptor. We have explored the theoretical consequences of this binding geometry for a CheR molecule associated with a cluster of chemotactic receptors. Calculations show that the CheR molecule will bind with high net affinity to the receptor lattice, having a high probability of being attached by one or both of its domains at any instant of time. Because of the relatively low affinity of its individual domains and the close proximity of neighboring receptors, it is likely that when one domain unbinds it will reattach to the array before the other domain unbinds. Stochastic simulations show that the enzyme will move through the receptor cluster in a hand-over-hand fashion, like a gibbon swinging through the branches of a tree. We explore the possible consequences of this motion, which we term "molecular brachiation", for chemotactic adaptation and suggest that a similar mechanism may be operative in other large assemblies of protein molecules. PMID:11916840

  6. Dimerization of Cell-Adhesion Molecules Can Increase Their Binding Strength.

    PubMed

    Huang, Wenmao; Qin, Meng; Li, Ying; Cao, Yi; Wang, Wei

    2017-02-14

    Cell-adhesion molecules (CAMs) often exist as homodimers under physiological conditions. However, owing to steric hindrance, simultaneous binding of two ligands to the homodimers at the same location can hardly be satisfied, and the molecular mechanism underlying this natural design is still unknown. Here, we present a theoretical model to understand the rupture behavior of cell-adhesion bonds formed by multiple binding ligands with a single receptor. We found that the dissociation forces for the cell-adhesion bond could be greatly enhanced in comparison with the monomer case through a ligand rebinding and exchange mechanism. We also confirmed this prediction by measuring dimeric cRGD (cyclic Arg-Gly-Asp) unbinding from integrin (αvβ3) using atomic force microscopy-based single-molecule force spectroscopy. Our finding addresses the mechanism of increasing the binding strength of cell-adhesion bonds through dimerization at the single-molecule level, representing a key step toward the understanding of complicated cell-adhesion behaviors. Moreover, our results also highlight a wealth of opportunities to design mechanically stronger bioconjunctions for drug delivery, biolabeling, and surface modification.

  7. Calreticulin is a microbial-binding molecule with phagocytosis-enhancing capacity.

    PubMed

    Liu, Xuemei; Xu, Na; Zhang, Shicui

    2013-09-01

    Calreticulin (CRT) is a highly conserved calcium-binding protein mainly involved in directing proper conformation of proteins and controlling calcium level. Accumulating data also show that CRT is emerging as an immune-relevant molecule. In this study, we demonstrated that the CRT gene from the amphioxus Branchiostoma japonicum, named Bjcrt, consisted of a signal peptide, three domains (N-, P-, C-domains) and an ER retrieval signal sequence (KDEL), which appears to be the ancient form of vertebrate CRTs, and Bjcrt was expressed in a tissue-specific manner, with the most abundant expression in the notochord. We also demonstrated for the first time that the recombinant BjCRT (rBjCRT) was able to bind the Gram-negative bacterium Escherichia coli and the Gram-positive bacterium Staphylococcus aureus. Moreover, both BjCRT as well as human recombinant calreticulin were able to promote the phagocytosis of E. coli and S. aureus by sea bass macrophages. These results indicate that CRT is a microbial-binding molecule and possesses an ability to enhance phagocytosis, a novel function assigned to CRT, reenforcing the notion that CRT is an immune-relevant molecule associated with host immune responses.

  8. Single-Molecule Counting of Point Mutations by Transient DNA Binding

    NASA Astrophysics Data System (ADS)

    Su, Xin; Li, Lidan; Wang, Shanshan; Hao, Dandan; Wang, Lei; Yu, Changyuan

    2017-03-01

    High-confidence detection of point mutations is important for disease diagnosis and clinical practice. Hybridization probes are extensively used, but are hindered by their poor single-nucleotide selectivity. Shortening the length of DNA hybridization probes weakens the stability of the probe-target duplex, leading to transient binding between complementary sequences. The kinetics of probe-target binding events are highly dependent on the number of complementary base pairs. Here, we present a single-molecule assay for point mutation detection based on transient DNA binding and use of total internal reflection fluorescence microscopy. Statistical analysis of single-molecule kinetics enabled us to effectively discriminate between wild type DNA sequences and single-nucleotide variants at the single-molecule level. A higher single-nucleotide discrimination is achieved than in our previous work by optimizing the assay conditions, which is guided by statistical modeling of kinetics with a gamma distribution. The KRAS c.34 A mutation can be clearly differentiated from the wild type sequence (KRAS c.34 G) at a relative abundance as low as 0.01% mutant to WT. To demonstrate the feasibility of this method for analysis of clinically relevant biological samples, we used this technology to detect mutations in single-stranded DNA generated from asymmetric RT-PCR of mRNA from two cancer cell lines.

  9. Binding of atoms and stability of molecules in Hartree and Thomas-Fermi type theories

    SciTech Connect

    Catto, I.; Lions, P.L. )

    1993-01-01

    This paper is the third of a series devoted to the study of the binding of atoms, molecules and ions and of the stability of general molecular systems including molecular ions, in the context of Hartree and Thomas-Fermi type theories. For Thomas-Fermi-von Weizsaecker or Thomas-Fermi-Dirac-von Weizsaecker models, it is proven here that neutral systems can be bound and in view of the results shown in the preceding parts this yields the stability of arbitrary molecules (general neutral molecular systems). For the Hartree and Hartree-Fock models, it is proven that neutral planar systems can be bound and this yields the stability of arbitrary tetraatomic molecules for instance. Various variants and extensions are also considered. 24 refs.

  10. Binding affinity of a small molecule to an amorphous polymer in a solvent. Part 1: free energy of binding to a binding site.

    PubMed

    Chunsrivirot, Surasak; Diao, Ying; Trout, Bernhardt L

    2011-10-18

    Crystallization is commonly used in a separation and purification process in the production of a wide range of materials in various industries. In industry, crystallization usually starts with heterogeneous nucleation on a foreign surface. The complicated mechanism of heterogeneous nucleation is not well understood; however, we hypothesize that there might be a possible correlation between binding affinity to a surface and enhancement of nucleation. Recent studies show that amorphous polymers can be used to control crystallization, selectively produce pharmaceutical polymorphs, and discover novel pharmaceutical polymorphs. To investigate the possible correlation between the binding affinity of one molecule to key binding sites (local binding) and heterogeneous nucleation activity as well as the possibility of using this binding affinity to help guide the selection of polymers that promote heterogeneous nucleation, we computed the free energy of binding of aspirin to four nonporous cross-linked polymers in an ethanol-water 38 v% mixture. These cross-linked polymers are poly(4-acryloylmorpholine) (PAM), poly(2-carboxyethyl acrylate) (PCEA), poly(4-hydroxylbutyl acrylate) (PHBA), and polystyrene (PS); all of them were cross-linked with divinylbenzene (DVB). These systems were used because their heterogeneous nucleation activities are available in literature, and the ranking is PAM > PCEA > PHBA ≈ PS. We generated three independent surfaces for each polymer and computed the free energy of binding of aspirin to the best binding site that we found on each surface. The average free energies of binding to the best sites of PAM, PCEA, PHBA, and PS are -20.4 ± 1.0, -16.7 ± 1.0, -14.4 ± 1.1, and -13.6 ± 1.1 kcal/mol, respectively. We found that the trend of the magnitudes of the average free energies of binding to the best sites is PAM > PCEA > PHBA ≈ PS. This trend is very similar to that of heterogeneous nucleation activity. Our results suggest the importance of the

  11. Measuring p53 Binding to Single DNA Molecules in a Nanofluidic Device

    NASA Astrophysics Data System (ADS)

    Whelsky, Amber; Gonzalez, Nicholas, Jr.; Gal, Susannah; Levy, Stephen

    2012-02-01

    Protein-DNA binding is central to several important cellular processes, for instance, the transfer of genetic information into proteins. The p53 protein plays a central role in regulating several major cell cycle pathways, in part by binding to well-characterized DNA sequences in the promoters of specific genes. Recent studies show that the most common mutation to the protein occurs in the region responsible for its binding to DNA. We have fabricated slit-like nanofluidic devices that allow us to trap and stretch single molecules of DNA containing a known recognition sequence of p53. We use fluorescent microscopy to observe the diffusion of a single p53 protein as it searches for its DNA recognition site. We measure the reaction rates of binding to selected DNA sequences as well as the one-dimensional, non-sequence specific diffusion of p53 along a stretched DNA molecule as a function of salt concentration. The mechanism of facilitated diffusion attempts to explain how proteins seem able to find their DNA target sequences much more quickly than would be expected from three-dimensional diffusion alone. We compare the observed search mechanism used by normal and mutated p53 from cancer cells to predictions based on this theory.

  12. Three-dimensional model of a selective theophylline-binding RNA molecule

    SciTech Connect

    Tung, Chang-Shung; Oprea, T.I.; Hummer, G.; Garcia, A.E.

    1995-07-01

    We propose a three-dimensional (3D) model for an RNA molecule that selectively binds theophylline but not caffeine. This RNA, which was found using SELEX [Jenison, R.D., et al., Science (1994) 263:1425] is 10,000 times more specific for theophylline (Kd=320 nM) than for caffeine (Kd=3.5 mM), although the two ligands are identical except for a methyl group substituted at N7 (present only in caffeine). The binding affinity for ten xanthine-based ligands was used to derive a Comparative Molecular Field Analysis (CoMFA) model (R{sup 2} = 0.93 for 3 components, with cross-validated R{sup 2} of 0.73), using the SYBYL and GOLPE programs. A pharmacophoric map was generated to locate steric and electrostatic interactions between theophylline and the RNA binding site. This information was used to identify putative functional groups of the binding pocket and to generate distance constraints. Based on a model for the secondary structure (Jenison et al., idem), the 3D structure of this RNA was then generated using the following method: each helical region of the RNA molecule was treated as a rigid body; single-stranded loops with specific end-to-end distances were generated. The structures of RNA-xanthine complexes were studied using a modified Monte Carlo algorithm. The detailed structure of an RNA-ligand complex model, as well as possible explanations for the theophylline selectivity will be discussed.

  13. Binding of europium complex to polymerizable macrocyclic molecules and its optical properties

    NASA Astrophysics Data System (ADS)

    Hidayat, Rahmat; Sugihara, Okihiro; Tsuchimori, Masaaki; Kagami, Manabu; Nishikubo, Tadatomi; Kaino, Toshikuni

    2007-07-01

    Study on the incorporation of trivalent europium (Eu3+) complex into polymerizable macrocylic molecules, namely calixarene and calixresorcinarene derivatives, has been carried out. Broadening of hypersensitive luminescence peak was observed in solution and polymer containing a compound of europium complex and calixresorcinarene monomer, which is ascribed to ligand field splitting due to anisotropic local field at the Eu3+ ion site. From 1H NMR spectroscopy, it is found that the chemical shift originated from the moiety at the upper rim of calixresorcinarene is remarkably downfield shifted. Pseudocontact shift analysis suggests that binding between Eu3+ ion and calixresorcinarene monomer is formed at the Eu3+ second coordination sphere. These experimental results show that the binding does not alter nephelauxetic effect of the Eu3+ first coordination shell, but only slightly change coordination structure and symmetry. Nevertheless, the binding results in longer luminescence lifetime in comparison to the case of pure europium complex indicating significant reduction of non-radiative decay.

  14. Hydration in drug design. 3. Conserved water molecules at the ligand-binding sites of homologous proteins.

    PubMed

    Poornima, C S; Dean, P M

    1995-12-01

    Water molecules are known to play an important rôle in mediating protein-ligand interactions. If water molecules are conserved at the ligand-binding sites of homologous proteins, such a finding may suggest the structural importance of water molecules in ligand binding. Structurally conserved water molecules change the conventional definition of 'binding sites' by changing the shape and complementarity of these sites. Such conserved water molecules can be important for site-directed ligand/drug design. Therefore, five different sets of homologous protein/protein-ligand complexes have been examined to identify the conserved water molecules at the ligand-binding sites. Our analysis reveals that there are as many as 16 conserved water molecules at the FAD binding site of glutathione reductase between the crystal structures obtained from human and E. coli. In the remaining four sets of high-resolution crystal structures, 2-4 water molecules have been found to be conserved at the ligand-binding sites. The majority of these conserved water molecules are either bound in deep grooves at the protein-ligand interface or completely buried in cavities between the protein and the ligand. All these water molecules, conserved between the protein/protein-ligand complexes from different species, have identical or similar apolar and polar interactions in a given set. The site residues interacting with the conserved water molecules at the ligand-binding sites have been found to be highly conserved among proteins from different species; they are more conserved compared to the other site residues interacting with the ligand. These water molecules, in general, make multiple polar contacts with protein-site residues.

  15. A New Design Strategy and Diagnostic to Tailor the DNA-Binding Mechanism of Small Organic Molecules and Drugs.

    PubMed

    Doan, Phi; Pitter, Demar R G; Kocher, Andrea; Wilson, James N; Goodson, Theodore

    2016-11-18

    The classical model for DNA groove binding states that groove binding molecules should adopt a crescent shape that closely matches the helical groove of DNA. Here, we present a new design strategy that does not obey this classical model. The DNA-binding mechanism of small organic molecules was investigated by synthesizing and examining a series of novel compounds that bind with DNA. This study has led to the emergence of structure-property relationships for DNA-binding molecules and/or drugs, which reveals that the structure can be designed to either intercalate or groove bind with calf thymus dsDNA by modifying the electron acceptor properties of the central heterocyclic core. This suggests that the electron accepting abilities of the central core play a key role in the DNA-binding mechanism. These small molecules were characterized by steady-state and ultrafast nonlinear spectroscopies. Bioimaging experiments were performed in live cells to evaluate cellular uptake and localization of the novel small molecules. This report paves a new route for the design and development of small organic molecules, such as therapeutics, targeted at DNA as their performance and specificity is dependent on the DNA-binding mechanism.

  16. Binding of two DNA molecules by type II topoisomerases for decatenation

    PubMed Central

    Kumar, Rupesh; Riley, Jane E.; Parry, Damian; Bates, Andrew D.; Nagaraja, Valakunja

    2012-01-01

    Topoisomerases (topos) maintain DNA topology and influence DNA transaction processes by catalysing relaxation, supercoiling and decatenation reactions. In the cellular milieu, division of labour between different topos ensures topological homeostasis and control of central processes. In Escherichia coli, DNA gyrase is the principal enzyme that carries out negative supercoiling, while topo IV catalyses decatenation, relaxation and unknotting. DNA gyrase apparently has the daunting task of undertaking both the enzyme functions in mycobacteria, where topo IV is absent. We have shown previously that mycobacterial DNA gyrase is an efficient decatenase. Here, we demonstrate that the strong decatenation property of the enzyme is due to its ability to capture two DNA segments in trans. Topo IV, a strong dedicated decatenase of E. coli, also captures two distinct DNA molecules in a similar manner. In contrast, E. coli DNA gyrase, which is a poor decatenase, does not appear to be able to hold two different DNA molecules in a stable complex. The binding of a second DNA molecule to GyrB/ParE is inhibited by ATP and the non-hydrolysable analogue, AMPPNP, and by the substitution of a prominent positively charged residue in the GyrB N-terminal cavity, suggesting that this binding represents a potential T-segment positioned in the cavity. Thus, after the GyrA/ParC mediated initial DNA capture, GyrB/ParE would bind efficiently to a second DNA in trans to form a T-segment prior to nucleotide binding and closure of the gate during decatenation. PMID:22989710

  17. Water molecules inside protein structure affect binding of monosaccharides with HIV-1 antibody 2G12.

    PubMed

    Ueno-Noto, Kaori; Takano, Keiko

    2016-10-05

    Water molecules inside biomolecules constitute integral parts of their structure and participate in the functions of the proteins. Some of the X-ray crystallographic data are insufficient for analyzing a series of ligand-protein complexes in the same condition. We theoretically investigated antibody binding abilities of saccharide ligands and the effects of the inner water molecules of ligand-antibody complexes. Classical molecular dynamics and quantum chemical simulations using a model with possible water molecules inside the protein were performed with saccharide ligands and Human Immunodeficiency Virus 1 neutralizing antibody 2G12 complexes to estimate how inner water molecules of the protein affect the dynamics of the complexes as well as the ligand-antibody interaction. Our results indicate the fact that d-fructose's strong affinity to the antibody was partly due to the good retentiveness of solvent water molecules of the ligand and its stability of the ligand's conformation and relative position in the active site. © 2016 Wiley Periodicals, Inc.

  18. Binding affinity of a small molecule to an amorphous polymer in a solvent. Part 2: preferential binding to local sites on a surface.

    PubMed

    Chunsrivirot, Surasak; Santiso, Erik; Trout, Bernhardt L

    2011-10-18

    Crystallization, a separation and purification process, is commonly used to produce a wide range of materials in various industries, and it usually begins with heterogeneous nucleation on a foreign surface in industrial practice and most other circumstances. Recent studies show that amorphous polymeric substrates are useful in controlling crystallization and selectively producing pharmaceutical polymorphs. In our previous publication, we investigated the possible correlation of the binding affinity of one molecule to key binding sites (local binding), and the possibility of using this binding affinity to guide the selection of polymers promoting heterogeneous nucleation. The studied systems were aspirin binding to four nonporous cross-linked polymers in ethanol-water 38 v% mixture. Cross-linked with divinylbenzene (DVB), these polymers were poly(4-acryloylmorpholine) (PAM), poly(2-carboxyethyl acrylate) (PCEA), poly(4-hydroxylbutyl acrylate) (PHBA), and polystyrene (PS). We discovered that the trend of the magnitudes of the average free energies of binding to the best sites is very similar to that of heterogeneous nucleation activities. This Article aims to investigate whether or not local binding to key sites is the important variable to describe heterogeneous nucleation as opposed to the overall/average binding affinity of molecules to a surface, and to investigate the possibility of using the overall binding affinity to guide the selection of polymers. We used the polymer surfaces generated from our previous study to calculate the overall binding affinity of aspirin molecules to the surface as measured by the preferential interaction coefficients of aspirin (1 m) to these polymers. We discovered that the trend of the average preferential interaction coefficients does not correlate as well to that of heterogeneous nucleation activities as the free energies of binding to the best sites. We also computed the average numbers of aspirin molecules associated with the

  19. Legume receptors perceive the rhizobial lipochitin oligosaccharide signal molecules by direct binding

    PubMed Central

    Broghammer, Angelique; Krusell, Lene; Blaise, Mickaël; Sauer, Jørgen; Sullivan, John T.; Maolanon, Nicolai; Vinther, Maria; Lorentzen, Andrea; Madsen, Esben B.; Jensen, Knud J.; Roepstorff, Peter; Thirup, Søren; Ronson, Clive W.; Thygesen, Mikkel B.; Stougaard, Jens

    2012-01-01

    Lipochitin oligosaccharides called Nod factors function as primary rhizobial signal molecules triggering legumes to develop new plant organs: root nodules that host the bacteria as nitrogen-fixing bacteroids. Here, we show that the Lotus japonicus Nod factor receptor 5 (NFR5) and Nod factor receptor 1 (NFR1) bind Nod factor directly at high-affinity binding sites. Both receptor proteins were posttranslationally processed when expressed as fusion proteins and extracted from purified membrane fractions of Nicotiana benthamiana or Arabidopsis thaliana. The N-terminal signal peptides were cleaved, and NFR1 protein retained its in vitro kinase activity. Processing of NFR5 protein was characterized by determining the N-glycosylation patterns of the ectodomain. Two different glycan structures with identical composition, Man3XylFucGlcNAc4, were identified by mass spectrometry and located at amino acid positions N68 and N198. Receptor–ligand interaction was measured by using ligands that were labeled or immobilized by application of chemoselective chemistry at the anomeric center. High-affinity ligand binding was demonstrated with both solid-phase and free solution techniques. The Kd values obtained for Nod factor binding were in the nanomolar range and comparable to the concentration range sufficient for biological activity. Structure-dependent ligand specificity was shown by using chitin oligosaccharides. Taken together, our results suggest that ligand recognition through direct ligand binding is a key step in the receptor-mediated activation mechanism leading to root nodule development in legumes. PMID:22859506

  20. Structural insights into binding of small molecule inhibitors to Enhancer of Zeste Homolog 2

    NASA Astrophysics Data System (ADS)

    Kalinić, Marko; Zloh, Mire; Erić, Slavica

    2014-11-01

    Enhancer of Zeste Homolog 2 (EZH2) is a SET domain protein lysine methyltransferase (PKMT) which has recently emerged as a chemically tractable and therapeutically promising epigenetic target, evidenced by the discovery and characterization of potent and highly selective EZH2 inhibitors. However, no experimental structures of the inhibitors co-crystallized to EZH2 have been resolved, and the structural basis for their activity and selectivity remains unknown. Considering the need to minimize cross-reactivity between prospective PKMT inhibitors, much can be learned from understanding the molecular basis for selective inhibition of EZH2. Thus, to elucidate the binding of small-molecule inhibitors to EZH2, we have developed a model of its fully-formed cofactor binding site and used it to carry out molecular dynamics simulations of protein-ligand complexes, followed by molecular mechanics/generalized born surface area calculations. The obtained results are in good agreement with biochemical inhibition data and reflect the structure-activity relationships of known ligands. Our findings suggest that the variable and flexible post-SET domain plays an important role in inhibitor binding, allowing possibly distinct binding modes of inhibitors with only small variations in their structure. Insights from this study present a good basis for design of novel and optimization of existing compounds targeting the cofactor binding site of EZH2.

  1. Identification of a peptide sequence involved in homophilic binding in the neural cell adhesion molecule NCAM

    PubMed Central

    1992-01-01

    The neural cell adhesion molecule NCAM is capable of mediating cell- cell adhesion via homophilic interactions. In this study, three strategies have been combined to identify regions of NCAM that participate directly in NCAM-NCAM binding: analysis of domain deletion mutations, mapping of epitopes of monoclonal antibodies, and use of synthetic peptides to inhibit NCAM activity. Studies on L cells transfected with NCAM mutant cDNAs using cell aggregation and NCAM- covasphere binding assays indicate that the third immunoglobulin-like domain is involved in homophilic binding. The epitopes of four monoclonal antibodies that have been previously shown to affect cell- cell adhesion mediated by NCAM were also mapped to domain 3. Overlapping hexapeptides were synthesized on plastic pins and assayed for binding with these monoclonal antibodies. One of them (PP) reacted specifically with the sequence KYSFNY. Synthetic oligopeptides containing the PP epitope were potent and specific inhibitors of NCAM binding activity. A substratum containing immobilized peptide conjugates also exhibited adhesiveness for neural retinal cells. Cell attachment was specifically inhibited by peptides that contained the PP- epitope and by anti-NCAM univalent antibodies. The shortest active peptide has the sequence KYSFNYDGSE, suggesting that this site is directly involved in NCAM homophilic interaction. PMID:1380002

  2. Proteomic analysis of egg white heparin-binding proteins: towards the identification of natural antibacterial molecules.

    PubMed

    Guyot, Nicolas; Labas, Valérie; Harichaux, Grégoire; Chessé, Magali; Poirier, Jean-Claude; Nys, Yves; Réhault-Godbert, Sophie

    2016-06-13

    The chicken egg resists most environmental microbes suggesting that it potentially contains efficient antimicrobial molecules. Considering that some heparin-binding proteins in mammals are antibacterial, we investigated the presence and the antimicrobial activity of heparin-binding proteins from chicken egg white. Mass spectrometry analysis of the proteins recovered after heparin-affinity chromatography, revealed 20 proteins, including known antimicrobial proteins (avidin, lysozyme, TENP, ovalbumin-related protein X and avian bêta-defensin 11). The antibacterial activity of three new egg candidates (vitelline membrane outer layer protein 1, beta-microseminoprotein-like (LOC101750704) and pleiotrophin) was demonstrated against Listeria monocytogenes and/or Salmonella enterica Enteritidis. We showed that all these molecules share the property to inhibit bacterial growth through their heparin-binding domains. However, vitelline membrane outer layer 1 has additional specific structural features that can contribute to its antimicrobial potential. Moreover, we identified potential supplementary effectors of innate immunity including mucin 5B, E-selectin ligand 1, whey acidic protein 3, peptidyl prolyl isomerase B and retinoic acid receptor responder protein 2. These data support the concept of using heparin affinity combined to mass spectrometry to obtain an overview of the various effectors of innate immunity composing biological milieus, and to identify novel antimicrobial candidates of interest in the race for alternatives to antibiotics.

  3. Proteomic analysis of egg white heparin-binding proteins: towards the identification of natural antibacterial molecules

    PubMed Central

    Guyot, Nicolas; Labas, Valérie; Harichaux, Grégoire; Chessé, Magali; Poirier, Jean-Claude; Nys, Yves; Réhault-Godbert, Sophie

    2016-01-01

    The chicken egg resists most environmental microbes suggesting that it potentially contains efficient antimicrobial molecules. Considering that some heparin-binding proteins in mammals are antibacterial, we investigated the presence and the antimicrobial activity of heparin-binding proteins from chicken egg white. Mass spectrometry analysis of the proteins recovered after heparin-affinity chromatography, revealed 20 proteins, including known antimicrobial proteins (avidin, lysozyme, TENP, ovalbumin-related protein X and avian bêta-defensin 11). The antibacterial activity of three new egg candidates (vitelline membrane outer layer protein 1, beta-microseminoprotein-like (LOC101750704) and pleiotrophin) was demonstrated against Listeria monocytogenes and/or Salmonella enterica Enteritidis. We showed that all these molecules share the property to inhibit bacterial growth through their heparin-binding domains. However, vitelline membrane outer layer 1 has additional specific structural features that can contribute to its antimicrobial potential. Moreover, we identified potential supplementary effectors of innate immunity including mucin 5B, E-selectin ligand 1, whey acidic protein 3, peptidyl prolyl isomerase B and retinoic acid receptor responder protein 2. These data support the concept of using heparin affinity combined to mass spectrometry to obtain an overview of the various effectors of innate immunity composing biological milieus, and to identify novel antimicrobial candidates of interest in the race for alternatives to antibiotics. PMID:27294500

  4. Effective tight-binding models for excitons in branched conjugated molecules

    NASA Astrophysics Data System (ADS)

    Li, Hao; Malinin, Sergey V.; Tretiak, Sergei; Chernyak, Vladimir Y.

    2013-08-01

    Effective tight-binding models have been introduced to describe vertical electronic excitations in branched conjugated molecules. The excited-state electronic structure is characterized by quantum particles (excitons) that reside on an irregular lattice (graph) that reflects the molecular structure. The methodology allows for the exciton spectra and energy-dependent exciton scattering matrices to be described in terms of a small number of lattice parameters which can be obtained from quantum-chemical computations using the exciton scattering approach as a tool. We illustrate the tight-binding model approach using the time-dependent Hartree-Fock computations in phenylacetylene oligomers. The on-site energies and hopping constants have been identified from the exciton dispersion and scattering matrices. In particular, resonant, as well as bound states, are reproduced for a symmetric quadruple branching center. The capability of the tight-binding model approach to describe the exciton-phonon coupling and energetic disorder in large branched conjugated molecules is briefly discussed.

  5. Discovery and Characterization of a Cell-Permeable, Small-Molecule c-Abl Kinase Activator that Binds to the Myristoyl Binding Site

    SciTech Connect

    Yang, Jingsong; Campobasso, Nino; Biju, Mangatt P.; Fisher, Kelly; Pan, Xiao-Qing; Cottom, Josh; Galbraith, Sarah; Ho, Thau; Zhang, Hong; Hong, Xuan; Ward, Paris; Hofmann, Glenn; Siegfried, Brett; Zappacosta, Francesca; Washio, Yoshiaki; Cao, Ping; Qu, Junya; Bertrand, Sophie; Wang, Da-Yuan; Head, Martha S.; Li, Hu; Moores, Sheri; Lai, Zhihong; Johanson, Kyung; Burton, George; Erickson-Miller, Connie; Simpson, Graham; Tummino, Peter; Copeland, Robert A.; Oliff, Allen

    2014-10-02

    c-Abl kinase activity is regulated by a unique mechanism involving the formation of an autoinhibited conformation in which the N-terminal myristoyl group binds intramolecularly to the myristoyl binding site on the kinase domain and induces the bending of the {alpha}I helix that creates a docking surface for the SH2 domain. Here, we report a small-molecule c-Abl activator, DPH, that displays potent enzymatic and cellular activity in stimulating c-Abl activation. Structural analyses indicate that DPH binds to the myristoyl binding site and prevents the formation of the bent conformation of the {alpha}I helix through steric hindrance, a mode of action distinct from the previously identified allosteric c-Abl inhibitor, GNF-2, that also binds to the myristoyl binding site. DPH represents the first cell-permeable, small-molecule tool compound for c-Abl activation.

  6. Small Molecules Engage Hot Spots through Cooperative Binding To Inhibit a Tight Protein-Protein Interaction.

    PubMed

    Liu, Degang; Xu, David; Liu, Min; Knabe, William Eric; Yuan, Cai; Zhou, Donghui; Huang, Mingdong; Meroueh, Samy O

    2017-03-28

    Protein-protein interactions drive every aspect of cell signaling, yet only a few small-molecule inhibitors of these interactions exist. Despite our ability to identify critical residues known as hot spots, little is known about how to effectively engage them to disrupt protein-protein interactions. Here, we take advantage of the ease of preparation and stability of pyrrolinone 1, a small-molecule inhibitor of the tight interaction between the urokinase receptor (uPAR) and its binding partner, the urokinase-type plasminogen activator uPA, to synthesize more than 40 derivatives and explore their effect on the protein-protein interaction. We report the crystal structure of uPAR bound to previously discovered pyrazole 3 and to pyrrolinone 12. While both 3 and 12 bind to uPAR and compete with a fluorescently labeled peptide probe, only 12 and its derivatives inhibit the full uPAR·uPA interaction. Compounds 3 and 12 mimic and engage different hot-spot residues on uPA and uPAR, respectively. Interestingly, 12 is involved in a π-cation interaction with Arg-53, which is not considered a hot spot. Explicit-solvent molecular dynamics simulations reveal that 3 and 12 exhibit dramatically different correlations of motion with residues on uPAR. Free energy calculations for the wild-type and mutant uPAR bound to uPA or 12 show that Arg-53 interacts with uPA or with 12 in a highly cooperative manner, thereby altering the contributions of hot spots to uPAR binding. The direct engagement of peripheral residues not considered hot spots through π-cation or salt-bridge interactions could provide new opportunities for enhanced small-molecule engagement of hot spots to disrupt challenging protein-protein interactions.

  7. Diatomic molecules and metallic adhesion, cohesion, and chemisorption - A single binding-energy relation

    NASA Technical Reports Server (NTRS)

    Ferrante, J.; Smith, J. R.; Rose, J. H.

    1983-01-01

    Potential-energy relations involving a few parameters in simple analytic forms have been found to represent well the energetics of a wide variety of diatomic molecules. However, such two-atom potential functions are not appropriate for metals. It is well known that, in the case of metals, there exist strong volume-dependent forces which can never be expressed as pairwise interactions. The present investigation has the objective to show that, in spite of the observation concerning metals, a single binding-energy relation can be found which accurately describes diatomic molecules as well as adhesion, cohesion, and chemisorption on metals. This universality reveals a commonality between the molecular and metallic bond.

  8. RIM - binding proteins (RBPs) couple Rab3 - interacting molecules (RIMs) to voltage - gated Ca2+ channels

    PubMed Central

    Hibino, H.; Pironkova, R.; Onwumere, O.; Vologodskaia, M.; Hudspeth, A. J.; Lesage, F.

    2007-01-01

    Summary Ca2+ influx through voltage-gated channels initiates the exocytotic fusion of synaptic vesicles to the plasma membrane. Here we show that RIM-binding proteins (RBPs), which associate with Ca2+ channels in hair cells, photoreceptors, and neurons, interact with α1D (L-type) and α1B (N-type) Ca2+-channel subunits. RBPs contain three Src homology 3 domains that bind to proline-rich motifs in α1 subunits and Rab3-interacting molecules (RIMs). Overexpression in PC12 cells of fusion proteins that suppress the interactions of RBPs with RIMs and α1 augments the exocytosis triggered by depolarization. RBPs may regulate the strength of synaptic transmission by creating a functional link between the synaptic-vesicle tethering apparatus, which includes RIMs and Rab3, and the fusion machinery, which includes Ca2+ channels and the SNARE complex. PMID:11988172

  9. Expression levels of MHC class I molecules are inversely correlated with promiscuity of peptide binding

    PubMed Central

    Chappell, Paul E; Meziane, El Kahina; Harrison, Michael; Magiera, Łukasz; Hermann, Clemens; Mears, Laura; Wrobel, Antoni G; Durant, Charlotte; Nielsen, Lise Lotte; Buus, Søren; Ternette, Nicola; Mwangi, William; Butter, Colin; Nair, Venugopal; Ahyee, Trudy; Duggleby, Richard; Madrigal, Alejandro; Roversi, Pietro; Lea, Susan M; Kaufman, Jim

    2015-01-01

    Highly polymorphic major histocompatibility complex (MHC) molecules are at the heart of adaptive immune responses, playing crucial roles in many kinds of disease and in vaccination. We report that breadth of peptide presentation and level of cell surface expression of class I molecules are inversely correlated in both chickens and humans. This relationship correlates with protective responses against infectious pathogens including Marek's disease virus leading to lethal tumours in chickens and human immunodeficiency virus infection progressing to AIDS in humans. We propose that differences in peptide binding repertoire define two groups of MHC class I molecules strategically evolved as generalists and specialists for different modes of pathogen resistance. We suggest that differences in cell surface expression level ensure the development of optimal peripheral T cell responses. The inverse relationship of peptide repertoire and expression is evidently a fundamental property of MHC molecules, with ramifications extending beyond immunology and medicine to evolutionary biology and conservation. DOI: http://dx.doi.org/10.7554/eLife.05345.001 PMID:25860507

  10. Properties of complexes formed by Na(+), Mg(2+), and Fe(2+) binding with benzene molecules.

    PubMed

    Kolakkandy, Sujitha; Pratihar, Subha; Aquino, Adelia J A; Wang, Hai; Hase, William L

    2014-10-09

    A theoretical investigation was performed to study cation-π interactions in complexes of benzene (Bz) with cations, that is, M(z+)(Bz)n for M(z+) = Na(+), Mg(2+), Fe(2+) and n = 1-3, using MP2 theory with the 6-31+G* and 6-311++G** basis sets and the DFT/(B3LYP and B3LYP-D)/6-311++G** methods. Binding energies and structures of the complexes are reported. The splitting between the quintet and single states of the Fe(2+) complexes was found to depend on the number of benzene molecules in the complex and the complex's structure. All of the M(z+)(Bz) complexes prefer a half-sandwich geometry. A geometry with the cation sandwiched between the two benzene rings was found for the M(z+)(Bz)2 complexes, with the benzene rings either in an eclipsed or staggered conformation. An approximate cyclic structure, with the cation at its center, was found for three benzene molecules interacting with the cation. The cation-benzene binding energy is substantial and equal to 22, 108, and 151 kcal/mol for the Na(+)(Bz), Mg(2+)(Bz), and Fe(2+)(Bz) complexes, respectively. The strength of the interaction of the cation with an individual benzene molecule decreases as the number of benzene molecules bound to the cation increases; for example, it is 108 kcal/mol for Mg(2+)(Bz), but only 71 kcal/mol for Mg(2+)(Bz)3. There is a range of values for the M(z+)(Bz)n intermolecular vibrational frequencies; for example, they are ∼230-360 and ∼10-330 cm(-1) for the Mg(2+)(Bz) and Mg(2+)(Bz)3 complexes, respectively. Binding of the cation to benzene both red and blue shifts the benzene vibrational frequencies. This shifting is larger for the Mg(2+) and Fe(2+) complexes, as compared to those for Na(+), as a result of the former's stronger cation-benzene binding. The present study is an initial step to understand the possible importance of cation-π interactions for polycyclic aromatic hydrocarbon aggregation processes during soot formation.

  11. Design of sequence-specific DNA binding molecules for DNA methyltransferase inhibition.

    PubMed

    Kang, JeenJoo S; Meier, Jordan L; Dervan, Peter B

    2014-03-05

    The CpG dyad, an important genomic feature in DNA methylation and transcriptional regulation, is an attractive target for small molecules. To assess the utility of minor groove binding oligomers for CpG recognition, we screened a small library of hairpin pyrrole-imidazole polyamides targeting the sequence 5'-CGCG-3' and assessed their sequence specificity using an unbiased next-generation sequencing assay. Our findings indicate that hairpin polyamide of sequence PyImβIm-γ-PyImβIm (1), previously identified as a high affinity 5'-CGCG-3' binder, favors 5'-GCGC-3' in an unanticipated reverse binding orientation. Replacement of one β alanine with Py to afford PyImPyIm-γ-PyImβIm (3) restores the preference for 5'-CGCG-3' binding in a forward orientation. The minor groove binding hairpin 3 inhibits DNA methyltransferase activity in the major groove at its target site more effectively than 1, providing a molecular basis for design of sequence-specific antagonists of CpG methylation.

  12. The effect of non-binding molecules on the gelation of HbS.

    PubMed

    Benedict, R C; Fall, L; Gill, S J; Hedlund, B

    1981-06-01

    The influence of an inert globular macromolecule upon the solubility of sickle cell hemoglobin has been determined as a function of the degree of oxygenation. The thermodynamic theory required to treat this and related problems is derived starting with the Gibbs-Duhem equation and introducing the effect of specific binding (oxygen) by use of the binding partition function. The treatment includes non-ideal solution behaviour as measured by osmotic pressure of highly concentrated macromolecular solutions. Application of the theoretical equation demonstrates how the solubility of hemoglobin is influenced by the presence of the binding ligand (oxygen) and the inert macromolecule, bovine serum albumin (BSA). Good agreement is obtained between experimentally determined and theoretically calculated solubilities using 1) oxygen binding curves to solution and gel phases, 2) activity coefficients from osmotic pressure data, 3) one solubility under the condition where oxygen and BSA are absent, and 4) the value of the water content of the gel phase. Examination of theoretical equations suggests that inert molecules of intermediate size, that are partially excluded from crystalline or gel phases, have the potential of generally increasing the solubility when non-ideal solution effects are small.

  13. Single-Molecule Studies of the Linker Histone H1 Binding to DNA and the Nucleosome.

    PubMed

    Yue, Hongjun; Fang, He; Wei, Sijie; Hayes, Jeffrey J; Lee, Tae-Hee

    2016-04-12

    Linker histone H1 regulates chromatin structure and gene expression. Investigating the dynamics and stoichiometry of binding of H1 to DNA and the nucleosome is crucial to elucidating its functions. Because of the abundant positive charges and the strong self-affinity of H1, quantitative in vitro studies of its binding to DNA and the nucleosome have generated results that vary widely and, therefore, should be interpreted in a system specific manner. We sought to overcome this limitation by developing a specially passivated microscope slide surface to monitor binding of H1 to DNA and the nucleosome at a single-molecule level. According to our measurements, the stoichiometry of binding of H1 to DNA and the nucleosome is very heterogeneous with a wide distribution whose averages are in reasonable agreement with previously published values. Our study also revealed that H1 does not dissociate from DNA or the nucleosome on a time scale of tens of minutes. We found that histone chaperone Nap1 readily dissociates H1 from DNA and superstoichiometrically bound H1 from the nucleosome, supporting a hypothesis whereby histone chaperones contribute to the regulation of the H1 profile in chromatin.

  14. Design of Sequence-Specific DNA Binding Molecules for DNA Methyltransferase Inhibition

    PubMed Central

    2015-01-01

    The CpG dyad, an important genomic feature in DNA methylation and transcriptional regulation, is an attractive target for small molecules. To assess the utility of minor groove binding oligomers for CpG recognition, we screened a small library of hairpin pyrrole-imidazole polyamides targeting the sequence 5′-CGCG-3′ and assessed their sequence specificity using an unbiased next-generation sequencing assay. Our findings indicate that hairpin polyamide of sequence PyImβIm-γ-PyImβIm (1), previously identified as a high affinity 5′-CGCG-3′ binder, favors 5′-GCGC-3′ in an unanticipated reverse binding orientation. Replacement of one β alanine with Py to afford PyImPyIm-γ-PyImβIm (3) restores the preference for 5′-CGCG-3′ binding in a forward orientation. The minor groove binding hairpin 3 inhibits DNA methyltransferase activity in the major groove at its target site more effectively than 1, providing a molecular basis for design of sequence-specific antagonists of CpG methylation. PMID:24502234

  15. Probing the ATP-Binding Pocket of Protein Kinase DYRK1A with Benzothiazole Fragment Molecules.

    PubMed

    Rothweiler, Ulli; Stensen, Wenche; Brandsdal, Bjørn Olav; Isaksson, Johan; Leeson, Frederick Alan; Engh, Richard Alan; Svendsen, John S Mjøen

    2016-11-10

    DYRK1A has emerged as a potential target for therapies of Alzheimer's disease using small molecules. On the basis of the observation of selective DYRK1A inhibition by firefly d-luciferin, we have explored static and dynamic structural properties of fragment sized variants of the benzothiazole scaffold with respect to DYRK1A using X-ray crystallography and NMR techniques. The compounds have excellent ligand efficiencies and show a remarkable diversity of binding modes in dynamic equilibrium. Binding geometries are determined in part by interactions often considered "weak", including "orthogonal multipolar" types represented by, for example, F-CO, sulfur-aromatic, and halogen-aromatic interactions, together with hydrogen bonds that are modulated by variation of electron withdrawing groups. These studies show how the benzothiazole scaffold is highly promising for the development of therapeutic DYRK1A inhibitors. In addition, the subtleties of the binding interactions, including dynamics, show how full structural studies are required to fully interpret the essential physical determinants of binding.

  16. Single-Molecule Imaging of an in Vitro-Evolved RNA Aptamer Reveals Homogeneous Ligand Binding Kinetics

    PubMed Central

    2009-01-01

    Many studies of RNA folding and catalysis have revealed conformational heterogeneity, metastable folding intermediates, and long-lived states with distinct catalytic activities. We have developed a single-molecule imaging approach for investigating the functional heterogeneity of in vitro-evolved RNA aptamers. Monitoring the association of fluorescently labeled ligands with individual RNA aptamer molecules has allowed us to record binding events over the course of multiple days, thus providing sufficient statistics to quantitatively define the kinetic properties at the single-molecule level. The ligand binding kinetics of the highly optimized RNA aptamer studied here displays a remarkable degree of uniformity and lack of memory. Such homogeneous behavior is quite different from the heterogeneity seen in previous single-molecule studies of naturally derived RNA and protein enzymes. The single-molecule methods we describe may be of use in analyzing the distribution of functional molecules in heterogeneous evolving populations or even in unselected samples of random sequences. PMID:19572753

  17. Localization of Bacillus thuringiensis Cry1A toxin-binding molecules in gypsy moth larval gut sections using fluorescence microscopy

    Treesearch

    Algimantas P. Valaitis

    2011-01-01

    The microbial insecticide Bacillus thuringiensis (Bt) produces Cry toxins, proteins that bind to the brush border membranes of gut epithelial cells of insects that ingest it, disrupting the integrity of the membranes, and leading to cell lysis and insect death. In gypsy moth, Lymantria dispar, two toxin-binding molecules for the...

  18. Telomerase Activity Detection with Amplification-Free Single Molecule Stochastic Binding Assay.

    PubMed

    Su, Xin; Li, Zehao; Yan, Xinzhong; Wang, Lei; Zhou, Xu; Wei, Lin; Xiao, Lehui; Yu, Changyuan

    2017-03-21

    Because the elongation of telomeres has been associated with tumorigenesis, it is of great interest to develop rapid and high-confidence telomerase activity detection methods for disease diagnosis. Currently, amplification-based strategies have been extensively explored for telomerase detection in vitro and in vivo. However, amplification is typically associated with poor reproducibility and high background, which hamper further applications of the strategies, particularly for real sample assays. Here, we demonstrate a new amplification-free single molecule imaging method for telomerase activity detection in vitro based on nucleic acid stochastic binding with total internal reflection fluorescence microscopy. The dynamic stochastic binding of a short fluorescent DNA probe with a genuine target yields a distinct kinetic signature from the background noise, allowing us to identify telomerase reaction products (TRPs) at the single molecule level. A limit-of-detection as low as 0.5 fM and a dynamic range of 0.5-500 fM for TRP detection were readily achieved. With this method, telomerase extracted from cancer cells was determined with sensitivity down to 10 cells. Moreover, the length distribution of TRPs was also determined by multiple stochastic probing, which could provide deep insight into the mechanistic study of telomerase catalysis.

  19. General approach for engineering small-molecule-binding DNA split aptamers.

    PubMed

    Kent, Alexandra D; Spiropulos, Nicholas G; Heemstra, Jennifer M

    2013-10-15

    Here we report a general method for engineering three-way junction DNA aptamers into split aptamers. Split aptamers show significant potential for use as recognition elements in biosensing applications, but reliable methods for generating these sequences are currently lacking. We hypothesize that the three-way junction is a "privileged architecture" for the elaboration of aptamers into split aptamers, as it provides two potential splitting sites that are distal from the target binding pocket. We propose a general method for split aptamer engineering that involves removing one loop region, then systematically modifying the number of base pairs in the remaining stem regions in order to achieve selective assembly only in the presence of the target small molecule. We screen putative split aptamer sequence pairs using split aptamer proximity ligation (StAPL) technology developed by our laboratory, but we validate that the results obtained using StAPL translate directly to systems in which the aptamer fragments are assembling noncovalently. We introduce four new split aptamer sequences, which triples the number of small-molecule-binding DNA split aptamers reported to date, and the methods described herein provide a reliable route for the engineering of additional split aptamers, dramatically advancing the potential substrate scope of DNA assembly based biosensors.

  20. Two and three-body interatomic dispersion energy contributions to binding in molecules and solids.

    SciTech Connect

    von Lilienfeld-Toal, Otto Anatole; Tkatchenko, Alexandre

    2010-03-01

    We present numerical estimates of the leading two- and three-body dispersion energy terms in van der Waals interactions for a broad variety of molecules and solids. The calculations are based on London and Axilrod-Teller-Muto expressions where the required interatomic dispersion energy coefficients, C{sub 6} and C{sub 9}, are computed 'on the fly' from the electron density. Inter- and intramolecular energy contributions are obtained using the Tang-Toennies (TT) damping function for short interatomic distances. The TT range parameters are equally extracted on the fly from the electron density using their linear relationship to van der Waals radii. This relationship is empiricially determined for all the combinations of He-Xe rare gas dimers, as well as for the He and Ar trimers. The investigated systems include the S22 database of noncovalent interactions, Ar, benzene and ice crystals, bilayer graphene, C{sub 60} dimer, a peptide (Ala{sub 10}), an intercalated drug-DNA model [ellipticine-d(CG){sub 2}], 42 DNA base pairs, a protein (DHFR, 2616 atoms), double stranded DNA (1905 atoms), and 12 molecular crystal polymorphs from crystal structure prediction blind test studies. The two- and three-body interatomic dispersion energies are found to contribute significantly to binding and cohesive energies, for bilayer graphene the latter reaches 50% of experimentally derived binding energy. These results suggest that interatomic three-body dispersion potentials should be accounted for in atomistic simulations when modeling bulky molecules or condensed phase systems.

  1. Multiple unfolding pathways of leucine binding protein (LBP) probed by single-molecule force spectroscopy (SMFS).

    PubMed

    Kotamarthi, Hema Chandra; Sharma, Riddhi; Narayan, Satya; Ray, Sayoni; Ainavarapu, Sri Rama Koti

    2013-10-02

    Experimental studies on the folding and unfolding of large multi-domain proteins are challenging, given the proteins' complex energy landscapes with multiple intermediates. Here, we report a mechanical unfolding study of a 346-residue, two-domain leucine binding protein (LBP) from the bacterial periplasm. Forced unfolding of LBP is a prerequisite for its translocation across the cytoplasmic membrane into the bacterial periplasm. During the mechanical stretching of LBP, we observe that 38% of the unfolding flux followed a two-state pathway, giving rise to a single unfolding force peak at ~70 pN with an unfolding contour length of 120 nm in constant-velocity single-molecule pulling experiments. The remaining 62% of the unfolding flux followed multiple three-state pathways, with intermediates having unfolding contour lengths in the range ~20-85 nm. These results suggest that the energy landscape of LBP is complex, with multiple intermediate states, and a large fraction of molecules go through intermediate states during the unfolding process. Furthermore, the presence of the ligand leucine increased the unfolding flux through the two-state pathway from 38% to 65%, indicating the influence of ligand binding on the energy landscape. This study suggests that unfolding through parallel pathways might be a general mechanism for the large two-domain proteins that are translocated to the bacterial periplasmic space.

  2. Combining Solvent Thermodynamic Profiles with Functionality Maps of the Hsp90 Binding Site to Predict the Displacement of Water Molecules

    PubMed Central

    2013-01-01

    Intermolecular interactions in the aqueous phase must compete with the interactions between the two binding partners and their solvating water molecules. In biological systems, water molecules in protein binding sites cluster at well-defined hydration sites and can form strong hydrogen-bonding interactions with backbone and side-chain atoms. Displacement of such water molecules is only favorable when the ligand can form strong compensating hydrogen bonds. Conversely, water molecules in hydrophobic regions of protein binding sites make only weak interactions, and the requirements for favorable displacement are less stringent. The propensity of water molecules for displacement can be identified using inhomogeneous fluid solvation theory (IFST), a statistical mechanical method that decomposes the solvation free energy of a solute into the contributions from different spatial regions and identifies potential binding hotspots. In this study, we employed IFST to study the displacement of water molecules from the ATP binding site of Hsp90, using a test set of 103 ligands. The predicted contribution of a hydration site to the hydration free energy was found to correlate well with the observed displacement. Additionally, we investigated if this correlation could be improved by using the energetic scores of favorable probe groups binding at the location of hydration sites, derived from a multiple copy simultaneous search (MCSS) method. The probe binding scores were not highly predictive of the observed displacement and did not improve the predictivity when used in combination with IFST-based hydration free energies. The results show that IFST alone can be used to reliably predict the observed displacement of water molecules in Hsp90. However, MCSS can augment IFST calculations by suggesting which functional groups should be used to replace highly displaceable water molecules. Such an approach could be very useful in improving the hit-to-lead process for new drug targets

  3. Combining solvent thermodynamic profiles with functionality maps of the Hsp90 binding site to predict the displacement of water molecules.

    PubMed

    Haider, Kamran; Huggins, David J

    2013-10-28

    Intermolecular interactions in the aqueous phase must compete with the interactions between the two binding partners and their solvating water molecules. In biological systems, water molecules in protein binding sites cluster at well-defined hydration sites and can form strong hydrogen-bonding interactions with backbone and side-chain atoms. Displacement of such water molecules is only favorable when the ligand can form strong compensating hydrogen bonds. Conversely, water molecules in hydrophobic regions of protein binding sites make only weak interactions, and the requirements for favorable displacement are less stringent. The propensity of water molecules for displacement can be identified using inhomogeneous fluid solvation theory (IFST), a statistical mechanical method that decomposes the solvation free energy of a solute into the contributions from different spatial regions and identifies potential binding hotspots. In this study, we employed IFST to study the displacement of water molecules from the ATP binding site of Hsp90, using a test set of 103 ligands. The predicted contribution of a hydration site to the hydration free energy was found to correlate well with the observed displacement. Additionally, we investigated if this correlation could be improved by using the energetic scores of favorable probe groups binding at the location of hydration sites, derived from a multiple copy simultaneous search (MCSS) method. The probe binding scores were not highly predictive of the observed displacement and did not improve the predictivity when used in combination with IFST-based hydration free energies. The results show that IFST alone can be used to reliably predict the observed displacement of water molecules in Hsp90. However, MCSS can augment IFST calculations by suggesting which functional groups should be used to replace highly displaceable water molecules. Such an approach could be very useful in improving the hit-to-lead process for new drug targets.

  4. Universal tight binding model for chemical reactions in solution and at surfaces. I. Organic molecules

    SciTech Connect

    Sheppard, T. J.; Lozovoi, A. Y.; Kohanoff, J. J.; Pashov, D. L.; Paxton, A. T.

    2014-07-28

    As is now well established, a first order expansion of the Hohenberg–Kohn total energy density functional about a trial input density, namely, the Harris–Foulkes functional, can be used to rationalize a non self consistent tight binding model. If the expansion is taken to second order then the energy and electron density matrix need to be calculated self consistently and from this functional one can derive a charge self consistent tight binding theory. In this paper we have used this to describe a polarizable ion tight binding model which has the benefit of treating charge transfer in point multipoles. This admits a ready description of ionic polarizability and crystal field splitting. It is necessary in constructing such a model to find a number of parameters that mimic their more exact counterparts in the density functional theory. We describe in detail how this is done using a combination of intuition, exact analytical fitting, and a genetic optimization algorithm. Having obtained model parameters we show that this constitutes a transferable scheme that can be applied rather universally to small and medium sized organic molecules. We have shown that the model gives a good account of static structural and dynamic vibrational properties of a library of molecules, and finally we demonstrate the model's capability by showing a real time simulation of an enolization reaction in aqueous solution. In two subsequent papers, we show that the model is a great deal more general in that it will describe solvents and solid substrates and that therefore we have created a self consistent quantum mechanical scheme that may be applied to simulations in heterogeneous catalysis.

  5. Single-molecule imaging of transcription factor binding to DNA in live mammalian cells.

    PubMed

    Gebhardt, J Christof M; Suter, David M; Roy, Rahul; Zhao, Ziqing W; Chapman, Alec R; Basu, Srinjan; Maniatis, Tom; Xie, X Sunney

    2013-05-01

    Imaging single fluorescent proteins in living mammalian cells is challenged by out-of-focus fluorescence excitation. To reduce out-of-focus fluorescence we developed reflected light-sheet microscopy (RLSM), a fluorescence microscopy method allowing selective plane illumination throughout the nuclei of living mammalian cells. A thin light sheet parallel to the imaging plane and close to the sample surface is generated by reflecting an elliptical laser beam incident from the top by 90° with a small mirror. The thin light sheet allows for an increased signal-to-background ratio superior to that in previous illumination schemes and enables imaging of single fluorescent proteins with up to 100-Hz time resolution. We demonstrated the single-molecule sensitivity of RLSM by measuring the DNA-bound fraction of glucocorticoid receptor (GR) and determining the residence times on DNA of various oligomerization states and mutants of GR and estrogen receptor-α (ER), which permitted us to resolve different modes of DNA binding of GR. We demonstrated two-color single-molecule imaging by observing the spatiotemporal colocalization of two different protein pairs. Our single-molecule measurements and statistical analysis revealed dynamic properties of transcription factors.

  6. Benchmarking density functional tight binding models for barrier heights and reaction energetics of organic molecules.

    PubMed

    Gruden, Maja; Andjeklović, Ljubica; Jissy, Akkarapattiakal Kuriappan; Stepanović, Stepan; Zlatar, Matija; Cui, Qiang; Elstner, Marcus

    2017-09-30

    Density Functional Tight Binding (DFTB) models are two to three orders of magnitude faster than ab initio and Density Functional Theory (DFT) methods and therefore are particularly attractive in applications to large molecules and condensed phase systems. To establish the applicability of DFTB models to general chemical reactions, we conduct benchmark calculations for barrier heights and reaction energetics of organic molecules using existing databases and several new ones compiled in this study. Structures for the transition states and stable species have been fully optimized at the DFTB level, making it possible to characterize the reliability of DFTB models in a more thorough fashion compared to conducting single point energy calculations as done in previous benchmark studies. The encouraging results for the diverse sets of reactions studied here suggest that DFTB models, especially the most recent third-order version (DFTB3/3OB augmented with dispersion correction), in most cases provide satisfactory description of organic chemical reactions with accuracy almost comparable to popular DFT methods with large basis sets, although larger errors are also seen for certain cases. Therefore, DFTB models can be effective for mechanistic analysis (e.g., transition state search) of large (bio)molecules, especially when coupled with single point energy calculations at higher levels of theory. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  7. Designing a high performance, stable spectroscopic biosensor for the binding of large and small molecules.

    PubMed

    Gosselin, E; Vanden Eynde, J Jacques; Petit, A; Conti, J; De Coninck, J

    2017-12-15

    In the context of FTIR ATR-based sensors, the organic layer covering the ATR element has to be as stable as possible for optimal spectroscopic measurements. Previously, this self-assembled covering was considered stable after several hours under a PBS flux, probably due to a hydrophobic barrier, which prevents water penetration into the grafted network. Stability and reactivity, measured simultaneously using FTIR ATR, identify the limits of the previously used molecular construction. For the first time, surface etching of the previous functionalised Ge devices (Ge-PEG-NHS), a few minutes after BSA injection, was observed. It was concluded that the molecular chain deformation of Ge-PEG-NHS likely occurred when large molecules were bound. BSA loaded onto a Ge-PEG-NHS surface led to network deprotection, with the probable disruption of hydrogen bonds for single barrier-based networks. This, in turn, was presumably influenced by the random deposition of the NHS moiety on the PEG chain. A new functionalised germanium device, using a rapid three-step in situ procedure, provides an efficient robust network composed of two protective barriers, ideal for the binding of various sized molecules. The Ge-APS-PEG-NHS device has shown exceptional sensitivity with regards to BSA and ethanolamine target molecules while offering homogeneous NHS distribution. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. Association of Catechin Molecules in Water: Quantitative Binding Study and Complex Structure Analysis.

    PubMed

    Ujihara, Tomomi; Hayashi, Nobuyuki

    2016-01-22

    Associations between catechin molecules were investigated by (1)H NMR titration experiments. Eight green tea catechins formed self-assembled dimers in water, and gallate-type catechins had a greater tendency to self-associate than non-gallate-type catechins. All eight catechins also associated as 1:1 heterodimer complexes. Investigation of complex formation of epigallocatechin-3-O-gallate (EGCg) and epigallocatechin (EGC) with the other catechins showed that the affinity between EGCg and 2,3-trans-gallate-type catechins was remarkably high, and the binding affinity of EGCg for ECg was also rather strong. In contrast, the non-gallate-type catechin EGC exhibited generally low binding affinity for other catechins. Structural analyses of the complexes by ROESY experiments and density functional theory calculations demonstrated that the higher binding abilities of gallate-type catechins are due to providing multiple intermolecular interactions that remain effective in an aqueous environment, such as aromatic/aromatic or CH/π interactions.

  9. The effect on structural and solvent water molecules of substrate binding to ferric horseradish peroxidase.

    PubMed

    Simpson, Niall; Adamczyk, Katrin; Hithell, Gordon; Shaw, Daniel J; Greetham, Gregory M; Towrie, Michael; Parker, Anthony W; Hunt, Neil T

    2015-01-01

    Ultrafast, multi-dimensional infrared spectroscopy, in the form of 2D-IR and pump-probe measurements, has been employed to investigate the effect of substrate binding on the structural dynamics of the horseradish peroxidase (HRP) enzyme. Using nitric oxide bound to the ferric haem of HRP as a sensitive probe of local dynamics, we report measurements of the frequency fluctuations (spectral diffusion) and vibrational lifetime of the NO stretching mode with benzohydroxamic acid (BHA) located in the substrate-binding position at the periphery of the haem pocket, in both D2O and H2O solvents. The results reveal that, with BHA bound to the enzyme, the local structural dynamics are insensitive to H/D exchange. These results are in stark contrast to those found in studies of the substrate-free enzyme, which demonstrated that the local chemical and dynamic environment of the haem ligand is influenced by water molecules. In light of the large changes in solvent accessibility caused by substrate binding, we discuss the potential for varying roles for the solvent in the haem pocket of HRP at different stages along the reaction coordinate of the enzymatic mechanism.

  10. The new generation drug candidate molecules: Spectral, electrochemical, DNA-binding and anticancer activity properties

    NASA Astrophysics Data System (ADS)

    Gölcü, Ayşegül; Muslu, Harun; Kılıçaslan, Derya; Çeşme, Mustafa; Eren, Özge; Ataş, Fatma; Demirtaş, İbrahim

    2016-09-01

    The new generation drug candidate molecules [Cu(5-Fu)2Cl2H2O] (NGDCM1) and [Zn(5-Fu)2(CH3COO)2] (NGDCM2) were obtained from the reaction of copper(II) and zinc(II) salts with the anticancer drug 5-fluoracil (5-Fu). These compounds have been characterized by spectroscopic and analytical techniques. Thermal behavior of the compounds were also investigated. The electrochemical properties of the compounds have been investigated by cyclic voltammetry (CV) using glassy carbon electrode. The biological activity of the NGDCM1 and NGDCM2 has been evaluated by examining their ability to bind to fish sperm double strand DNA (FSdsDNA) with UV spectroscopy. UV studies of the interaction of the 5-Fu and metal derivatives with FSdsDNA have shown that these compounds can bind to FSdsDNA. The binding constants of the compounds with FSdsDNA have also been calculated. Thermal decomposition of the compounds lead to the formation of CuO and ZnO as final products. The effect of proliferation 5-Fu, NGDCM1 and NGDCM2 were examined on the HeLa cells using real-time cell analyzer with three different concentrations.

  11. Predictive DFT +U Methods for Small Molecule Binding in MOF-74

    NASA Astrophysics Data System (ADS)

    Mann, Gregory; Lee, Kyuho; Cococcioni, Matteo; Smit, Berend; Neaton, Jeffrey

    2015-03-01

    In order to use density functional theory (DFT) to reliably treat small molecule binding at open metal sites in metal-organic frameworks (MOFs), electron correlation effects associated with the localized d-states present at the metal centers must be accounted for. Incorporation of a Hubbard U-like term can be an approximate but computationally efficient means, yielding excellent agreement with experiment provided an appropriate value for the parameter U is chosen. To predict adsorption energetics for as-yet unsynthesized MOFs, we would need to select U using a systematic, physically motivated approach rather than the ad hoc methods typically employed. Here, we use an ab initiolinear response approach to calculate U. We show that U values determined with this method reproduce previous results for the binding of carbon dioxide in Co-MOF-74 and Cu-MOF-74, and we discuss the method's application to other 3d metals in theMOF-74 framework; our preliminary results suggest that a wide range of U's above a critical value will produce accurate binding energies. Finally, we present U values calculated for Co2 + ions in other systems, probing the environment dependence of this parameter. This work supported by DOE, and computational resources provided by NERSC.

  12. Applications of Engineered DNA-Binding Molecules Such as TAL Proteins and the CRISPR/Cas System in Biology Research

    PubMed Central

    Fujita, Toshitsugu; Fujii, Hodaka

    2015-01-01

    Engineered DNA-binding molecules such as transcription activator-like effector (TAL or TALE) proteins and the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) (CRISPR/Cas) system have been used extensively for genome editing in cells of various types and species. The sequence-specific DNA-binding activities of these engineered DNA-binding molecules can also be utilized for other purposes, such as transcriptional activation, transcriptional repression, chromatin modification, visualization of genomic regions, and isolation of chromatin in a locus-specific manner. In this review, we describe applications of these engineered DNA-binding molecules for biological purposes other than genome editing. PMID:26404236

  13. Functional analysis of glyco-molecules that bind with influenza virus.

    PubMed

    Takahashi, Tadanobu

    2016-01-01

    Influenza A virus (IAV) recognizes terminal sialic acid of sialoglyco-conjugates on host cells through the viral envelope glycoprotein hemagglutinin (HA), followed by initiation of entry into the cells. Molecular species of sialic acid are largely divided into two moieties: N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc). A receptor for IAV infection generally means Neu5Ac. Almost all equine IAVs and some human, swine, and duck IAVs bind not only to Neu5Ac but also to Neu5Gc. In nonhuman animals, Neu5Gc has been detected in swine and equine tracheas and the duck colon, which are the main replication sites of mammalian and avian IAVs. Therefore, Neu5Gc in these sites has been suggested to be a functional receptor for IAV infection. Humans cannot synthesize Neu5Gc due to a genetic defect of the Neu5Gc-synthesizing enzyme. We evaluated the receptor function of Neu5Gc in IAV infection in human cells. Our results indicated that Neu5Gc expression on the surface of human cells is not a functional receptor for IAV infection and that it has a negative effect on infectivity of IAV possessing Neu5Gc binding ability. IAV also binds to non-sialo 3-O-sulfated galactosylceramide (sulfatide). Sulfatide has been suggested to be a functional receptor for IAV infection. However, we have shown that sulfatide is not a functional receptor for IAV infection and that the binding of HA with sulfatide enhances progeny virus production. It is expected that functions of these glyco-molecules can be used in prevention and development of new drugs against IAV.

  14. Soluble form of the endothelial adhesion molecule CD146 binds preferentially CD16+ monocytes.

    PubMed

    Garibaldi, Silvano; Barisione, Chiara; Ghigliotti, Giorgio; Spallarossa, Paolo; Barsotti, Antonio; Fabbi, Patrizia; Corsiglia, Luca; Palmieri, Daniela; Palombo, Domenico; Brunelli, Claudio

    2012-06-01

    The cell adhesion molecule CD146 is normally located at the endothelial cell-to-cell junction and colocalizes with actin cytoskeleton. The soluble form of CD146 (sCD146) has been identified in the endothelial cell supernatant and in normal human plasma, and is increased in pathologic conditions with altered endothelial function. Soluble CD146 binding to monocytes promotes their transendothelial migration, which represents a central step in the development of atherosclerotic plaque. Since peripheral blood monocytes are characterized by a phenotypic and functional heterogeneity, with different transendothelial migration capacity, we hypothesized that monocyte subsets differently bind sCD146. Based on surface CD14 and CD16 expression monocytes were distinguished by flow cytometry (FACS) into three subsets: CD14++/CD16-, CD14++/CD16+ and CD14+/CD16+. CD16+ monocytes have been found to possess higher transendothelial migration ability. FACS analysis on blood monocytes from 30 healthy subjects revealed that higher percentages of CD14++/CD16+ (median, first and third quartile: 2.26, 1.62-3.87) and of CD14+/CD16+ (2.59, 1.28-4.80) were positive for CD146 (both p < 0.01), in comparison to CD14++/CD16- (0.66, 0.47-1.01). Moreover, in vitro treatment of ficoll separated monocytes with recombinant CD146 showed that both CD16+ subsets increased their percentage of CD146-positive events compared to CD16- monocytes (p < 0.01). Soluble CD146 levels were evaluated by ELISA in plasma samples of subjects from our study group and showed a correlation with percentage of CD146-positive CD14+/CD16+ monocyte subset. In this work we have demonstrated that monocyte subsets behave differently with regard to their sCD146 binding activity; because binding of CD146 influences transendothelial migration of monocytes, modulation of monocyte-CD146 interaction may represent a potential target to limit atherosclerotic plaque development.

  15. Measuring binding kinetics of aromatic thiolated molecules with nanoparticles via surface-enhanced Raman spectroscopy

    NASA Astrophysics Data System (ADS)

    Devetter, Brent M.; Mukherjee, Prabuddha; Murphy, Catherine J.; Bhargava, Rohit

    2015-05-01

    Colloidal plasmonic nanomaterials, consisting of metals such as gold and silver, are excellent candidates for advanced optical probes and devices, but precise control over surface chemistry is essential for realizing their full potential. Coupling thiolated (R-SH) molecules to nanoprobe surfaces is a convenient and established route to tailor surface properties. The ability to dynamically probe and monitor the surface chemistry of nanoparticles in solution is essential for rapidly manufacturing spectroscopically tunable nanoparticles. In this study, we report the development of surface-enhanced Raman spectroscopy (SERS) as a method to monitor the kinetics of gold-thiolate bond formation on colloidal gold nanoparticles. A theoretical model combining SERS enhancement with the Beer-Lambert law is proposed to explain ensemble scattering and absorption effects in colloids during chemisorption. In order to maximize biological relevance and signal reproducibility, experiments used to validate the model focused on maintaining nanoparticle stability after the addition of water-soluble aromatic thiolated molecules. Our results indicate that ligand exchange on gold nanoparticles follow a first-order Langmuir adsorption model with rate constants on the order of 0.01 min-1. This study demonstrates an experimental spectroscopic method and theoretical model for monitoring binding kinetics that may prove useful for designing novel probes.Colloidal plasmonic nanomaterials, consisting of metals such as gold and silver, are excellent candidates for advanced optical probes and devices, but precise control over surface chemistry is essential for realizing their full potential. Coupling thiolated (R-SH) molecules to nanoprobe surfaces is a convenient and established route to tailor surface properties. The ability to dynamically probe and monitor the surface chemistry of nanoparticles in solution is essential for rapidly manufacturing spectroscopically tunable nanoparticles. In this

  16. A Switchable Open/closed Polyaromatic Macrocycle that Shows Reversible Binding of Long Hydrophilic Molecules.

    PubMed

    Kurihara, Kohei; Yazaki, Kohei; Akita, Munetaka; Yoshizawa, Michito

    2017-09-11

    In spite of wide-ranging previous studies on synthetic macrocycles, the installation of open-close functions into the frameworks remains a challenge. We present a new polyaromatic macrocycle capable of switching between open and closed forms in response to external stimuli, namely, base and acid. The macrocycle, which is prepared in three steps, has a well-defined hydrophobic cavity with a length of around 1 nm, surrounded by four pH-responsive acridinium panels. The open and closed structures were confirmed by single-crystal X-ray analysis. The cylindrical cavity can bind long hydrophilic molecules up to 2.7 nm in length in neutral water and then release the bound guests through a reversible open-to-closed structural change upon simple addition of base. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. HLA-B37 and HLA-A2.1 molecules bind largely nonoverlapping sets of peptides.

    PubMed Central

    Carreno, B M; Anderson, R W; Coligan, J E; Biddison, W E

    1990-01-01

    T-cell recognition of peptides that are bound and presented by class I major histocompatibility complex molecules is highly specific. At present it is unclear what role class I peptide binding plays relative to T-cell receptor specificity in determination of immune recognition. A previous study from our group demonstrated that the HLA-A2.1 molecule could bind to 25% of the members of a panel of unrelated synthetic peptides as assessed by a functional peptide competition assay. To determine the peptide-binding specificity of another HLA class I molecule, we have examined the capacity of this panel of peptides to compete for the presentation of influenza virus nucleoprotein peptide NP-(335-350) by HLA-B37 to NP-peptide-specific HLA-B37-restricted cytotoxic T-lymphocyte lines. Forty-two percent of peptides tested were capable of inhibiting NP-(335-350) presentation by HLA-B37. Remarkably, none of these HLA-B37-binding peptides belong to the subset that was previously shown to bind to the HLA-A2.1 molecule. Only the NP-(335-350) peptide was capable of binding to both HLA-A2.1 and HLA-B37. These findings demonstrate that the peptide-binding specificities of HLA-B37 and HLA-A2.1 are largely nonoverlapping and suggest that, from the universe of peptides, individual HLA class I molecules can bind to clearly distinct subsets of these peptides. PMID:2333291

  18. Recombinant glycoproteins that inhibit complement activation and also bind the selectin adhesion molecules.

    PubMed

    Rittershaus, C W; Thomas, L J; Miller, D P; Picard, M D; Geoghegan-Barek, K M; Scesney, S M; Henry, L D; Sen, A C; Bertino, A M; Hannig, G; Adari, H; Mealey, R A; Gosselin, M L; Couto, M; Hayman, E G; Levin, J L; Reinhold, V N; Marsh, H C

    1999-04-16

    Soluble human complement receptor type 1 (sCR1, TP10) has been expressed in Chinese hamster ovary (CHO) DUKX-B11 cells and shown to inhibit the classical and alternative complement pathways in vitro and in vivo. A truncated version of sCR1 lacking the long homologous repeat-A domain (LHR-A) containing the C4b binding site has similarly been expressed and designated sCR1[desLHR-A]. sCR1[desLHR-A] was shown to be a selective inhibitor of the alternative complement pathway in vitro and to function in vivo. In this study, sCR1 and sCR1[desLHR-A] were expressed in CHO LEC11 cells with an active alpha(1,3)-fucosyltransferase, which makes possible the biosynthesis of the sialyl-Lewisx (sLex) tetrasaccharide (NeuNAcalpha2-3Galbeta1-4(Fucalpha1-3)GlcNAc) during post-translational glycosylation. The resulting glycoproteins, designated sCR1sLex and sCR1[desLHR-A]sLex, respectively, retained the complement regulatory activities of their DUKX B11 counterparts, which lack alpha(1-3)-fucose. Carbohydrate analysis of purified sCR1sLex and sCR1[desLHR-A]sLex indicated an average incorporation of 10 and 8 mol of sLex/mol of glycoprotein, respectively. sLex is a carbohydrate ligand for the selectin adhesion molecules. sCR1sLex was shown to specifically bind CHO cells expressing cell surface E-selectin. sCR1[desLHR-A]sLex inhibited the binding of the monocytic cell line U937 to human aortic endothelial cells, which had been activated with tumor necrosis factor-alpha to up-regulate the expression of E-selectin. sCR1sLex inhibited the binding of U937 cells to surface-adsorbed P-selectin-IgG. sCR1sLex and sCR1[desLHR-A]sLex have thus demonstrated both complement regulatory activity and the capacity to bind selectins and to inhibit selectin-mediated cell adhesion in vitro.

  19. Single Molecule Imaging of Transcription Factor Binding to DNA in Live Mammalian Cells

    PubMed Central

    Gebhardt, J Christof M; Suter, David M; Roy, Rahul; Zhao, Ziqing W; Chapman, Alec R; Basu, Srinjan; Maniatis, Tom; Xie, X Sunney

    2013-01-01

    Imaging single fluorescent proteins in living mammalian cells is challenging due to out-of-focus fluorescence excitation by common microscopy schemes. We report the development of a novel fluorescence microscopy method, reflected light sheet microscopy (RLSM), which allows selective plane illumination throughout the nucleus of living mammalian cells, for reducing out-of-focus fluorescence signal. Generation of a thin light sheet parallel to the imaging plane and close to the sample surface is achieved by reflecting an elliptical laser beam incident from the top by 45° with a small mirror. The thin light sheet allows for an increased signal-to-background ratio superior to previous illumination schemes and enables imaging of single fluorescent proteins with up to 100 Hz time resolution. We demonstrate the sensitivity of RLSM by measuring the DNA-bound fraction of glucocorticoid receptor (GR) and determine the residence times on DNA of various oligomerization states and mutants of GR and estrogen receptor (ER), enabling us to resolve different modes of DNA binding of GR. Finally, we demonstrate two-color single molecule imaging by observing the spatio-temporal co-localization of two different protein pairs. The combination of our single molecule measurements and statistical analysis reveals dynamic properties of transcription factors in live mammalian cells. PMID:23524394

  20. Measuring binding kinetics of aromatic thiolated molecules with nanoparticles via surface-enhanced Raman spectroscopy

    PubMed Central

    DeVetter, Brent M.; Mukherjee, Prabuddha; Murphy, Catherine J.; Bhargava, Rohit

    2015-01-01

    Colloidal plasmonic nanomaterials, consisting of metals such as gold and silver, are excellent candidates for advanced optical probes and devices, but precise control over surface chemistry is essential for realizing their full potential. Coupling thiolated (R-SH) molecules to nanoprobe surfaces is a convenient and established route to tailor surface properties. The ability to dynamically probe and monitor the surface chemistry of nanoparticles in solution is essential for rapidly manufacturing spectroscopically tunable nanoparticles. In this study, we report the development of surface-enhanced Raman spectroscopy (SERS) as a method to monitor the kinetics of gold-thiolate bond formation on colloidal gold nanoparticles. A theoretical model combining SERS enhancement with the Beer-Lambert law is proposed to explain ensemble scattering and absorption effects in colloids during chemisorption. In order to maximize biological relevance and signal reproducibility, experiments used to validate the model focused on maintaining nanoparticle stability after the addition of water-soluble aromatic thiolated molecules. Our results indicate that ligand exchange on gold nanoparticles follow a first-order Langmuir adsorption model with rate constants on the order of 0.01 min 1. This study demonstrates an experimental spectroscopic method and theoretical model for monitoring binding kinetics that may prove useful for designing novel probes. PMID:25905515

  1. A Small Molecule Bidentate-Binding Dual Inhibitor Probe of the LRRK2 and JNK Kinases

    PubMed Central

    Feng, Yangbo; Chambers, Jeremy W.; Iqbal, Sarah; Koenig, Marcel; Park, HaJeung; Cherry, Lisa; Hernandez, Pamela; Figuera-Losada, Mariana; LoGrasso, Philip V.

    2013-01-01

    Both JNK and LRRK2 are associated with Parkinson’s disease (PD). Here we report a reasonably selective and potent kinase inhibitor (compound 6) that bound to both JNK and LRRK2 (a dual inhibitor). A bidentate-binding strategy that simultaneously utilized the ATP hinge binding and a unique protein surface site outside of the ATP pocket was applied to the design and identification of this kind of inhibitor. Compound 6 was a potent JNK3 and modest LRRK2 dual inhibitor with an enzyme IC50 value of 12 nM and 99 nM (LRRK2-G2019S), respectively. 6 also exhibited good cell potency, inhibited LRRK2:G2019S induced mitochondrial dysfunction in SHSY5Y cells, and was demonstrated to be reasonably selective against a panel of 116 kinases from representative kinase families. Design of such a probe molecule may help enable testing if dual JNK and LRRK2 inhibitions have added or synergistic efficacy in protecting against neurodegeneration in PD. PMID:23751758

  2. Detection of metal binding sites on functional S-layer nanoarrays using single molecule force spectroscopy.

    PubMed

    Tang, Jilin; Ebner, Andreas; Kraxberger, Bernhard; Leitner, Michael; Hykollari, Alba; Kepplinger, Christian; Grunwald, Christian; Gruber, Hermann J; Tampé, Robert; Sleytr, Uwe B; Ilk, Nicola; Hinterdorfer, Peter

    2009-10-01

    Crystalline bacterial cell surface layers (S-layers) show the ability to recrystallize into highly regular pattern on solid supports. In this study, the genetically modified S-layer protein SbpA of Lysinibacillus sphaericus CCM 2177, carrying a hexa-histidine tag (His(6)-tag) at the C-terminus, was used to generate functionalized two-dimensional nanoarrays on a silicon surface. Atomic force microscopy (AFM) was applied to explore the topography and the functionality of the fused His(6)-tags. The accessibility of the His(6)-tags was demonstrated by in-situ anti-His-tag antibody binding to the functional S-layer array. The metal binding properties of the His(6)-tag was investigated by single molecule force microscopy. For this purpose, newly developed tris-NTA was tethered to the AFM tips via a flexible polyethylene glycol (PEG) linker. The functionalized tips showed specific interactions with S-layer containing His(6)-tags in the presence of nickel ions. Thus the His(6)-tag is located at the outer surface of the S-layer and can be used for stable but reversible attachment of functional tris-NTA derivatives.

  3. Acceleration of protein backbone NMR assignment by combinatorial labeling: Application to a small molecule binding study.

    PubMed

    Hein, Christopher; Löhr, Frank; Schwarz, Daniel; Dötsch, Volker

    2017-05-01

    Selective labeling with stable isotopes has long been recognized as a valuable tool in protein NMR to alleviate signal overlap and sensitivity limitations. In this study, combinatorial (15) N-, (13) C(α) -, and (13) C'-selective labeling has been used during the backbone assignment of human cyclophilin D to explore binding of an inhibitor molecule. Using a cell-free expression system, a scheme that involves (15) N, 1-(13) C, 2-(13) C, fully (15) N/(13) C, and unlabeled amino acids was optimized to gain a maximum of assignment information from three samples. This scheme was combined with time-shared triple-resonance NMR experiments, which allows a fast and efficient backbone assignment by giving the unambiguous assignment of unique amino acid pairs in the protein, the identity of ambiguous pairs and information about all 19 non-proline amino acid types. It is therefore well suited for binding studies where de novo assignments of amide (1) H and (15) N resonances need to be obtained, even in cases where sensitivity is the limiting factor. © 2016 Wiley Periodicals, Inc.

  4. Small Molecule Inhibited Parathyroid Hormone Mediated cAMP Response by N–Terminal Peptide Binding

    PubMed Central

    Kumar, Amit; Baumann, Monika; Balbach, Jochen

    2016-01-01

    Ligand binding to certain classes of G protein coupled receptors (GPCRs) stimulates the rapid synthesis of cAMP through G protein. Human parathyroid hormone (PTH), a member of class B GPCRs, binds to its receptor via its N–terminal domain, thereby activating the pathway to this secondary messenger inside cells. Presently, GPCRs are the target of many pharmaceuticals however, these drugs target only a small fraction of structurally known GPCRs (about 10%). Coordination complexes are gaining interest due to their wide applications in the medicinal field. In the present studies we explored the potential of a coordination complex of Zn(II) and anthracenyl–terpyridine as a modulator of the parathyroid hormone response. Preferential interactions at the N–terminal domain of the peptide hormone were manifested by suppressed cAMP generation inside the cells. These observations contribute a regulatory component to the current GPCR–cAMP paradigm, where not the receptor itself, but the activating hormone is a target. To our knowledge, this is the first report about a coordination complex modulating GPCR activity at the level of deactivating its agonist. Developing such molecules might help in the control of pathogenic PTH function such as hyperparathyroidism, where control of excess hormonal activity is essentially required. PMID:26932583

  5. Small Molecule Recognition of c-Src via the Imatinib-Binding Conformation

    PubMed Central

    Dar, Arvin C.; Lopez, Michael S.; Shokat, Kevan M.

    2009-01-01

    Summary The cancer drug, Imatinib, is a selective Abl kinase inhibitor which does not inhibit the closely related kinase c-Src. This one drug and its ability to selectively inhibit Abl over c-Src has been a guiding principle in virtually all kinase drug discovery efforts in the last fifteen years. A prominent hypothesis explaining the selectivity of Imatinib is that Abl has an intrinsic ability to adopt an inactive conformation (termed DFG-out), whereas c-Src appears to pay a high intrinsic energetic penalty for adopting this conformation effectively excluding Imatinib from its ATP pocket. This explanation of the difference in binding affinity of Imatinib for Abl versus c-Src makes the striking prediction that it would not be possible to design an inhibitor that binds to the inactive conformation of c-Src with high affinity. We now report the discovery of a series of such inhibitors. We use structure-activity relationships and X-ray crystallography to confirm our findings. These studies suggest that small molecules are capable of inducing the generally unfavourable DFG-out conformation in c-Src. PMID:18940662

  6. Chick neural retina adhesion and survival molecule is a retinol-binding protein

    SciTech Connect

    Schubert, D.; LaCorbiere, M.; Esch, F.

    1986-01-01

    A 20,000-D protein called purpurin has recently been isolated from the growth-conditioned medium of cultured embryonic chick neural retina cells. Purpurin is a constituent of adherons and promotes cell-adheron adhesion by interacting with a cell surface heparan sulfate proteoglycan. It also prolongs the survival of cultured neural retina cells. This paper shows that purpurin is a secretory protein that has sequence homology with a human protein synthesized in the liver that transports retinol in the blood, the serum retinol-binding protein (RBP). Purpurin binds (/sup 3/H)retinol, and both purpurin and chick serum RBP stimulate the adhesion of neural retina cells, although the serum protein is less active than purpurin. Purpurin and the serum RBP are, however, different molecules, for the serum protein is approx.3.000 D larger than purpurin and has different silver-staining characteristics. Finally, purpurin supports the survival of dissociated ciliary ganglion cells, indicating that RBPs can act as ciliary neurotrophic factors.

  7. Computational and Functional Analyses of a Small-Molecule Binding Site in ROMK

    PubMed Central

    Swale, Daniel R.; Sheehan, Jonathan H.; Banerjee, Sreedatta; Husni, Afeef S.; Nguyen, Thuy T.; Meiler, Jens; Denton, Jerod S.

    2015-01-01

    The renal outer medullary potassium channel (ROMK, or Kir1.1, encoded by KCNJ1) critically regulates renal tubule electrolyte and water transport and hence blood volume and pressure. The discovery of loss-of-function mutations in KCNJ1 underlying renal salt and water wasting and lower blood pressure has sparked interest in developing new classes of antihypertensive diuretics targeting ROMK. The recent development of nanomolar-affinity small-molecule inhibitors of ROMK creates opportunities for exploring the chemical and physical basis of ligand-channel interactions required for selective ROMK inhibition. We previously reported that the bis-nitro-phenyl ROMK inhibitor VU591 exhibits voltage-dependent knock-off at hyperpolarizing potentials, suggesting that the binding site is located within the ion-conduction pore. In this study, comparative molecular modeling and in silico ligand docking were used to interrogate the full-length ROMK pore for energetically favorable VU591 binding sites. Cluster analysis of 2498 low-energy poses resulting from 9900 Monte Carlo docking trajectories on each of 10 conformationally distinct ROMK comparative homology models identified two putative binding sites in the transmembrane pore that were subsequently tested for a role in VU591-dependent inhibition using site-directed mutagenesis and patch-clamp electrophysiology. Introduction of mutations into the lower site had no effect on the sensitivity of the channel to VU591. In contrast, mutations of Val168 or Asn171 in the upper site, which are unique to ROMK within the Kir channel family, led to a dramatic reduction in VU591 sensitivity. This study highlights the utility of computational modeling for defining ligand-ROMK interactions and proposes a mechanism for inhibition of ROMK. PMID:25762321

  8. Potentiometric sensors doped with biomolecules as a new approach to small molecule/biomolecule binding kinetics analysis.

    PubMed

    Daems, D; De Wael, K; Vissenberg, K; Van Camp, G; Nagels, L

    2014-04-15

    The most successful binding kinetics analysis systems at this moment include surface plasmon resonance (SPR), quartz microcrystal balance (QMB) and surface acoustic wave (SAW). Although these are powerful methods, they generally are complex, expensive and require the use of monolayers. Here, we report on potentiometric sensors as an inexpensive and simple alternative to do binding kinetics analysis between small molecules in solution and biomolecules (covalently) attached in a biopolymer sensor coating layer. As an example, dopamine and an anti-dopamine aptamer were used as the small molecule and the biomolecule respectively. Binding between both follows a Langmuir adsorption type model and creates a surface potential. The system operates in Flow Injection Analysis mode (FIA). Besides being an interesting new binding kinetics tool, the approach allows systematic design of potentiometric biosensors (in the present study a dopamine sensor), and gives new insights into the functioning of ion-selective electrodes (ISE's).

  9. The influence of intramolecular sulfur-lone pair interactions on small-molecule drug design and receptor binding.

    PubMed

    Hudson, B M; Nguyen, E; Tantillo, D J

    2016-04-28

    Sulfur-lone pair interactions are important conformational control elements in sulfur-containing heterocycles that abound in pharmaceuticals, natural products, agrochemicals, polymers and other important classes of organic molecules. Nonetheless, the role of intramolecular sulfur-lone pair interactions in the binding of small molecules to receptors is often overlooked. Here we analyze the magnitudes and origins of these interactions for a variety of biologically relevant small molecules using quantum chemical and automated docking calculations. In most cases examined in this study, the lowest energy conformation of the small molecule displays a sulfur-lone pair close contact. However, docking studies, both published and new, often predict that conformations without sulfur-lone pair contacts have the best binding affinity for their respective receptors. This is a serious problem. Since many of these predicted bound conformations are not actually energetically accessible, pursuing design (e.g., drug design) around these binding modes necessarily will lead, serendipity aside, to dead end designs. Our results constitute a caution that one best not neglect these interactions when predicting the binding affinities of potential ligands (drugs or not) for hosts (enzymes, receptors, DNA, RNA, synthetic hosts). Moreover, a better understanding and awareness of sulfur-lone pair interactions should facilitate the rational modulation of host-guest interactions involving sulfur-containing molecules.

  10. Critical contribution of beta chain residue 57 in peptide binding ability of both HLA-DR and -DQ molecules.

    PubMed Central

    Nepom, B S; Nepom, G T; Coleman, M; Kwok, W W

    1996-01-01

    Position 57 in the beta chain of HLA class II molecules maintains an Asp/non-Asp dimorphism that has been conserved through evolution and is implicated in susceptibility to some autoimmune diseases. The latter effect may be due to the influence of this residue on the ability of class II alleles to bind specific pathogenic peptides. We utilized highly homologous pairs of both DR and DQ alleles that varied at residue 57 to investigate the impact of this dimorphism on binding of model peptides. Using a direct binding assay of biotinylated peptides on whole cells expressing the desired alleles, we report several peptides that bind differentially to the allele pairs depending on the presence or absence of Asp at position 57. Peptides with negatively charged residues at anchor position 9 bind well to alleles not containing Asp at position 57 in the beta chain but cannot bind well to homologous Asp-positive alleles. By changing the peptides at the single residue predicted to interact with this position 57, we demonstrate a drastically altered or reversed pattern of binding. Ala analog peptides confirm these interactions and identify a limited set of interaction sites between the bound peptides and the class II molecules. Clarification of the impact of specific class II polymorphisms on generating unique allele-specific peptide binding "repertoires" will aid in our understanding of the development of specific immune responses and HLA-associated diseases. PMID:8692969

  11. Electronic structure and binding geometry of tetraphenylporphyrin-derived molecules adsorbed on metal and metal oxide surfaces

    NASA Astrophysics Data System (ADS)

    Coh, Senia

    Tetraphenylporphyrin (TPP)-derived molecules have been studied extensively as efficient photosensitizers when chemisorbed on the metal oxide substrates in dye-sensitized solar cells. Still, many fundamental electronic properties of the dye/oxide interface are not understood and need careful consideration. In this thesis we present a comprehensive study of the electronic structure, energy level alignment and the adsorption geometry of the TPP-derived dye molecules adsorbed on TiO2(110), ZnO(1120) and Ag(100) single crystal surfaces using ultra-high vacuum (UHV) based surface sensitive techniques. The alignment of the molecular energy levels with respect to the TiO 2 and ZnO band edges for all TPP-derived molecules we studied was found to be insensitive to either the nature of the functional groups located on the phenyl rings, presence of zinc as a central metal ion and different binding geometry of the molecules. Binding geometry, molecule-molecule interaction and the aggregation effects in the adsorbed layer, that were observed in the UV-visible spectra of the molecules adsorbed on ZnO substrate were not observed in the ultraviolet photoemission (UPS) and inverse photoemission (IPS) spectra of the occupied and unoccupied molecular states. Using near edge X-ray absorption fine structure (NEXAFS) and scanning tunneling microscopy (STM), binding geometry of the two representative TPP-derivatives was directly determined to be upright, with the porphyrin ring under large angle with respect to the surface for the p-ZnTCPP molecules and with the porphyrin ring parallel to the surface for the m-ZnTCPP molecules. We observe that the energies and the energy level alignment of the ZnTPP molecular levels measured in UPS and IPS depend on the substrate on which the molecules are adsorbed (Ag(100) or TiO2(110) single crystal surfaces). The differences are attributed to different charge screening properties of these two materials. Image charges created in the substrates during

  12. Computational analysis of protein-protein interfaces involving an alpha helix: insights for terphenyl–like molecules binding

    PubMed Central

    2013-01-01

    Background Protein-Protein Interactions (PPIs) are key for many cellular processes. The characterization of PPI interfaces and the prediction of putative ligand binding sites and hot spot residues are essential to design efficient small-molecule modulators of PPI. Terphenyl and its derivatives are small organic molecules known to mimic one face of protein-binding alpha-helical peptides. In this work we focus on several PPIs mediated by alpha-helical peptides. Method We performed computational sequence- and structure-based analyses in order to evaluate several key physicochemical and surface properties of proteins known to interact with alpha-helical peptides and/or terphenyl and its derivatives. Results Sequence-based analysis revealed low sequence identity between some of the analyzed proteins binding alpha-helical peptides. Structure-based analysis was performed to calculate the volume, the fractal dimension roughness and the hydrophobicity of the binding regions. Besides the overall hydrophobic character of the binding pockets, some specificities were detected. We showed that the hydrophobicity is not uniformly distributed in different alpha-helix binding pockets that can help to identify key hydrophobic hot spots. Conclusions The presence of hydrophobic cavities at the protein surface with a more complex shape than the entire protein surface seems to be an important property related to the ability of proteins to bind alpha-helical peptides and low molecular weight mimetics. Characterization of similarities and specificities of PPI binding sites can be helpful for further development of small molecules targeting alpha-helix binding proteins. PMID:23768251

  13. Effects of pH and polysaccharides on peptide binding to class II major histocompatibility complex molecules.

    PubMed Central

    Harding, C V; Roof, R W; Allen, P M; Unanue, E R

    1991-01-01

    The binding of immunogenic peptides to class II major histocompatibility molecules was examined at various pH values. We studied binding of peptides containing residues 52-61 from hen egg lysozyme (HEL) to I-Ak on fixed peritoneal macrophages or to solubilized affinity-purified I-Ak. Optimum binding occurred at pH 5.5-6.0 with accelerated kinetics relative to pH 7.4; equilibrium binding was also higher at pH 5.5-6.0 than at 7.4. Similar enhancement at pH 5-6 was observed for the binding of hemoglobin-(64-76) to I-Ek and of ribonuclease-(41-61) to I-Ak. In contrast, the binding of HEL-(34-45) to I-Ak was minimally enhanced at acid pH. Dissociation of cell-associated or purified peptide-I-Ak complexes was minimal between pH 5.5 and 7.4, with increased dissociation only at or below pH 4.0 [HEL-(46-61)] or pH 5.0 [HEL-(34-45)]. Thus, optimum peptide binding occurs at pH values similar to the endosomal environment, where the complexes appear to be formed during antigen processing. In addition, we examined the effect of a number of polysaccharides on the binding of peptide to I-Ak. None of these competed with the HEL peptide 125I-labeled YE52-61 for binding to I-Ak. [3H]Dextran also failed to bind purified I-Ak. Polysaccharides do not appear to bind to class II major histocompatibility complex molecules, which explains the T-cell independence of polysaccharide antigens. PMID:2011583

  14. The binding energies of one and two water molecules to the first transition-row metal positive ions. II

    NASA Technical Reports Server (NTRS)

    Rosi, Marzio; Bauschlicher, Charles W., Jr.

    1990-01-01

    The present investigation of H2O's binding energy to transition-metal ions proceeds from the D(2h) structure and bends the two water molecules out of plane. The molecule is constrained to have C(2v) symmetry, so that each water molecule and metal ion lies on a plane. The ground states are bent only for Mn(H2O)2(+) and Zn(H2O)2(+), where only 4s4p hybridization is energetically favorable; 4s4p hybridization reduces repulsion.

  15. The binding energies of one and two water molecules to the first transition-row metal positive ions. II

    NASA Technical Reports Server (NTRS)

    Rosi, Marzio; Bauschlicher, Charles W., Jr.

    1990-01-01

    The present investigation of H2O's binding energy to transition-metal ions proceeds from the D(2h) structure and bends the two water molecules out of plane. The molecule is constrained to have C(2v) symmetry, so that each water molecule and metal ion lies on a plane. The ground states are bent only for Mn(H2O)2(+) and Zn(H2O)2(+), where only 4s4p hybridization is energetically favorable; 4s4p hybridization reduces repulsion.

  16. R-Ras Regulates Murine T Cell Migration and Intercellular Adhesion Molecule-1 Binding

    PubMed Central

    Yan, Xiaocai; Yan, Mingfei; Guo, Yihe; Singh, Gobind; Chen, Yuhong; Yu, Mei; Wang, Demin; Hillery, Cheryl A.; Chan, Andrew M.

    2015-01-01

    The trafficking of T-lymphocytes to peripheral draining lymph nodes is crucial for mounting an adaptive immune response. The role of chemokines in the activation of integrins via Ras-related small GTPases has been well established. R-Ras is a member of the Ras-subfamily of small guanosine-5’-triphosphate-binding proteins and its role in T cell trafficking has been investigated in R-Ras null mice (Rras−/−). An examination of the lymphoid organs of Rras−/− mice revealed a 40% reduction in the cellularity of the peripheral lymph nodes. Morphologically, the high endothelial venules of Rras−/− mice were more disorganized and less mature than those of wild-type mice. Furthermore, CD4+ and CD8+ T cells from Rras−/− mice had approximately 42% lower surface expression of L-selectin/CD62L. These aberrant peripheral lymph node phenotypes were associated with proliferative and trafficking defects in Rras−/− T cells. Furthermore, R-Ras could be activated by the chemokine, CCL21. Indeed, Rras−/− T cells had approximately 14.5% attenuation in binding to intercellular adhesion molecule 1 upon CCL21 stimulation. Finally, in a graft-versus host disease model, recipient mice that were transfused with Rras−/− T cells showed a significant reduction in disease severity when compared with mice transplanted with wild-type T cells. These findings implicate a role for R-Ras in T cell trafficking in the high endothelial venules during an effective immune response. PMID:26710069

  17. Geometrically centered region: a "wet" model of protein binding hot spots not excluding water molecules.

    PubMed

    Li, Zhenhua; Li, Jinyan

    2010-12-01

    A protein interface can be as "wet" as a protein surface in terms of the number of immobilized water molecules. This important water information has not been explicitly taken by computational methods to model and identify protein binding hot spots, overlooking the water role in forming interface hydrogen bonds and in filing cavities. Hot spot residues are usually clustered at the core of the protein binding interfaces. However, traditional machine learning methods often identify the hot spot residues individually, breaking the cooperativity of the energetic contribution. Our idea in this work is to explore the role of immobilized water and meanwhile to capture two essential properties of hot spots: the compactness in contact and the far distance from bulk solvent. Our model is named geometrically centered region (GCR). The detection of GCRs is based on novel tripartite graphs, and atom burial levels which are a concept more intuitive than SASA. Applying to a data set containing 355 mutations, we achieved an F measure of 0.6414 when ΔΔG ≥ 1.0 kcal/mol was used to define hot spots. This performance is better than Robetta, a benchmark method in the field. We found that all but only one of the GCRs contain water to a certain degree, and most of the outstanding hot spot residues have water-mediated contacts. If the water is excluded, the burial level values are poorly related to the ΔΔG, and the model loses its performance remarkably. We also presented a definition for the O-ring of a GCR as the set of immediate neighbors of the residues in the GCR. Comparative analysis between the O-rings and GCRs reveals that the newly defined O-ring is indeed energetically less important than the GCR hot spot, confirming a long-standing hypothesis. Copyright © 2010 Wiley-Liss, Inc.

  18. A Small Molecule Inhibits Akt through Direct Binding to Akt and Preventing Akt Membrane Translocation*

    PubMed Central

    Kim, Donghwa; Sun, Mei; He, Lili; Zhou, Qing-Hua; Chen, Jun; Sun, Xia-Meng; Bepler, Gerold; Sebti, Said M.; Cheng, Jin Q.

    2010-01-01

    The Akt pathway is frequently hyperactivated in human cancer and functions as a cardinal nodal point for transducing extracellular and intracellular oncogenic signals and, thus, presents an exciting target for molecular therapeutics. Here we report the identification of a small molecule Akt/protein kinase B inhibitor, API-1. Although API-1 is neither an ATP competitor nor substrate mimetic, it binds to pleckstrin homology domain of Akt and blocks Akt membrane translocation. Furthermore, API-1 treatment of cancer cells results in inhibition of the kinase activities and phosphorylation levels of the three members of the Akt family. In contrast, API-1 had no effects on the activities of the upstream Akt activators, phosphatidylinositol 3-kinase, phosphatidylinositol-dependent kinase-1, and mTORC2. Notably, the kinase activity and phosphorylation (e.g. Thr(P)308 and Ser(P)473) levels of constitutively active Akt, including a naturally occurring mutant AKT1-E17K, were inhibited by API-1. API-1 is selective for Akt and does not inhibit the activation of protein kinase C, serum and glucocorticoid-inducible kinase, protein kinase A, STAT3, ERK1/2, or JNK. The inhibition of Akt by API-1 resulted in induction of cell growth arrest and apoptosis selectively in human cancer cells that harbor constitutively activated Akt. Furthermore, API-1 inhibited tumor growth in nude mice of human cancer cells in which Akt is elevated but not of those cancer cells in which it is not. These data indicate that API-1 directly inhibits Akt through binding to the Akt pleckstrin homology domain and blocking Akt membrane translocation and that API-1 has anti-tumor activity in vitro and in vivo and could be a potential anti-cancer agent for patients whose tumors express hyperactivated Akt. PMID:20068047

  19. Combining conformational sampling and selection to identify the binding mode of zinc-bound amyloid peptides with bifunctional molecules

    NASA Astrophysics Data System (ADS)

    Xu, Liang; Gao, Ke; Bao, Chunyu; Wang, Xicheng

    2012-08-01

    The pathogenesis of Alzheimer's disease (AD) has been suggested to be related with the aggregation of amyloid β (Aβ) peptides. Metal ions (e.g. Cu, Fe, and Zn) are supposed to induce the aggregation of Aβ. Recent development of bifunctional molecules that are capable of interacting with Aβ and chelating biometal ions provides promising therapeutics to AD. However, the molecular mechanism for how Aβ, metal ions, and bifunctional molecules interact with each other is still elusive. In this study, the binding mode of Zn2+-bound Aβ with bifunctional molecules was investigated by the combination of conformational sampling of full-length Aβ peptides using replica exchange molecular dynamics simulations (REMD) and conformational selection using molecular docking and classical MD simulations. We demonstrate that Zn2+-bound Aβ(1-40) and Aβ(1-42) exhibit different conformational ensemble. Both Aβ peptides can adopt various conformations to recognize typical bifunctional molecules with different binding affinities. The bifunctional molecules exhibit their dual functions by first preferentially interfering with hydrophobic residues 17-21 and/or 30-35 of Zn2+-bound Aβ. Additional interactions with residues surrounding Zn2+ could possibly disrupt interactions between Zn2+ and Aβ, which then facilitate these small molecules to chelate Zn2+. The binding free energy calculations further demonstrate that the association of Aβ with bifunctional molecules is driven by enthalpy. Our results provide a feasible approach to understand the recognition mechanism of disordered proteins with small molecules, which could be helpful to the design of novel AD drugs.

  20. [99mTc(CO)3]+-(HE)3-ZIGF1R:4551, a new Affibody conjugate for visualization of insulin-like growth factor-1 receptor expression in malignant tumours.

    PubMed

    Orlova, Anna; Hofström, Camilla; Strand, Joanna; Varasteh, Zohreh; Sandstrom, Mattias; Andersson, Karl; Tolmachev, Vladimir; Gräslund, Torbjörn

    2013-02-01

    Radionuclide imaging of insulin-like growth factor type 1 receptor (IGF-1R) expression in tumours might be used for selection of patients who would benefit from IGF-1R-targeted therapy. We have previously shown the feasibility of IGF-1R imaging using the Affibody molecule (111)In-DOTA-His(6)-Z(IGF1R:4551). The use of (99m)Tc instead of (111)In should improve sensitivity and resolution of imaging, and reduce the dose burden to patients. We hypothesized that inclusion of a HEHEHE tag instead of a His(6) tag in Z(IGF1R:4551) would permit its convenient purification using IMAC, enable labelling with [(99m)Tc(CO)(3)](+), and improve its biodistribution. Z(IGF1R:4551) was expressed with a HEHEHE tag in the N terminus. The resulting (HE)(3)-Z(IGF1R:4551) construct was labelled with [(99m)Tc(CO)(3)](+). Targeting of IGF-1R-expressing cells using [(99m)Tc(CO)(3)](+)-(HE)(3)-Z(IGF1R:4551) was evaluated in vitro and in vivo. (HE)(3)-Z(IGF1R:4551) was stably labelled with (99m)Tc with preserved specific binding to IGF-1R-expressing DU-145 prostate cancer cells in vitro. In mice, [(99m)Tc(CO)(3)](+)-(HE)(3)-Z(IGF1R:4551) accumulated in IGF-1R-expressing organs (pancreas, stomach, lung and salivary gland). [(99m)Tc(CO)(3)](+)-(HE)(3)-Z(IGF1R:4551) demonstrated 3.6-fold lower accumulation in the liver and spleen than (111)In-DOTA-Z(IGF1R:4551). In NMRI nu/nu mice with DU-145 prostate cancer xenografts, the tumour uptake was 1.32 ± 0.11 %ID/g and the tumour-to-blood ratio was 4.4 ± 0.3 at 8 h after injection. The xenografts were visualized using a gamma camera 6 h after injection. (99m)Tc(CO)(3)](+)-(HE)(3)-Z(IGF1R:4551) is a promising candidate for visualization of IGF-1R expression in malignant tumours.

  1. Structural and functional characterization of full-length heparin-binding growth associated molecule.

    PubMed Central

    Hampton, B S; Marshak, D R; Burgess, W H

    1992-01-01

    Heparin-binding growth-associated molecule (HB-GAM) was purified from adult bovine brain and chicken heart. The yield of HB-GAM is increased by 5- to 10-fold when 250 mM NaCl is added to the homogenization buffer, indicating that HB-GAM may exist as a complex with an insoluble component of the tissue. The complete amino acid sequence of the brain-derived HB-GAM was established by automated Edman degradation of the intact protein and chemically or enzymatically derived fragments. The mass of bovine HB-GAM as determined by plasma desorption time-of-flight mass spectrometry is 15,291 mass units, which compares favorably with the calculated mass of 15,289 based on the amino acid sequence. Therefore, HB-GAM has not undergone any major post-translational modifications other than cleavage of the signal peptide. These results indicate that previous amino acid sequence analysis of this protein was carried out using truncated HB-GAM. Full-length HB-GAM is not a mitogen for Balb/3T3 clone A31, Balb MK, NRK, or human umbilical vein endothelial cells. HB-GAM does, however, have adhesive properties and neurite extension activity for chick embryo cerebral cortical derived neurons when presented to these cells as a substrate. HB-GAM had little neurite extension activity when presented as a soluble factor. Images PMID:1550956

  2. Determination of the equilibrium constant of C60 fullerene binding with drug molecules.

    PubMed

    Mosunov, Andrei A; Pashkova, Irina S; Sidorova, Maria; Pronozin, Artem; Lantushenko, Anastasia O; Prylutskyy, Yuriy I; Parkinson, John A; Evstigneev, Maxim P

    2017-03-01

    We report a new analytical method that allows the determination of the magnitude of the equilibrium constant of complexation, Kh, of small molecules to C60 fullerene in aqueous solution. The developed method is based on the up-scaled model of C60 fullerene-ligand complexation and contains the full set of equations needed to fit titration datasets arising from different experimental methods (UV-Vis spectroscopy, (1)H NMR spectroscopy, diffusion ordered NMR spectroscopy, DLS). The up-scaled model takes into consideration the specificity of C60 fullerene aggregation in aqueous solution and allows the highly dispersed nature of C60 fullerene cluster distribution to be accounted for. It also takes into consideration the complexity of fullerene-ligand dynamic equilibrium in solution, formed by various types of self- and hetero-complexes. These features make the suggested method superior to standard Langmuir-type analysis, the approach used to date for obtaining quantitative information on ligand binding with different nanoparticles.

  3. Heme binding site in apomyoglobin may be effectively targeted with small molecules to control aggregation.

    PubMed

    Azami-Movahed, Mehrnaz; Shariatizi, Sajad; Sabbaghian, Marjan; Ghasemi, Atiyeh; Ebrahim-Habibi, Azadeh; Nemat-Gorgani, Mohsen

    2013-02-01

    A number of ligands with affinities for the heme binding site of apomyoglobin were tested to control amorphous and fibrillar aggregation in the protein. Several techniques, including fluorescence, dynamic light scattering, transmission electron microscopy, dot blot analysis combined with viability studies were employed for structural characterization and cytotoxicity assessment of the intermediate and final protein structures formed during the aggregation process. Of the small molecules investigated, chrysin and Nile red with high structural similarities to heme were chosen for further studies. Only fibril formation was found to be prevented by Nile red, while chrysin, with a greater structural flexibility, was able to prevent both types of aggregate formation. The two ligands were found to influence aggregation at different stages of intermediate structure formation, an ability determined by their degrees of similarities with heme. Based on structural characterization and toxicity studies, it is concluded that ligands similar in structure to heme may be effective in influencing various stages of aggregate formation and toxicity potencies of the protein structures. Since metalloproteins constitute more than thirty percent of all known proteins, it is concluded that the present strategy may be of general significance.

  4. [Influence of Ureaplasma urealyticum infection on the sperm-egg binding associated molecule, sulfogalactosylglycerolipid].

    PubMed

    Wu, Tingting; Lu, Meige; Hu, Yanqin; Guo, Qiangsu; Xu, Chen

    2004-09-01

    To study the influence of Ureaplasma urealyticum (Uu) infection on the sperm-egg binding associated molecule, sulfogalactosylglycerolipid (SGG). Epididymal sperm was collected from adult mice. The sperm suspension was randomly divided into 4 groups: Uu group (coincubated with Uu suspension), medium group (coincubated with Uu medium), normal group and PRS group. The indirect immunofluorescence technique was used to localize SGG on the sperm membrane and to observe the influence of Uu on SGG. In the epididymal sperm, SGG was localized to the head plasma membrane overlaying the acrosomal region. The SGG-positive rate of the sperm coincubated with Uu medium was 82.0%, while that of the sperm coincubated with Uu suspension was reduced to 39.0% (P = 0.001). Uu can adhere to the sperm surface. SGG might be a membrane receptor on the sperm surface for Uu infection of the mammalian male genital tract. The blockage of SGG by Uu might be one of the molecular mechanisms correlative to male infertility induced by Uu infection.

  5. A novel binding protein of single immunoglobulin IL-1 receptor-related molecule: Paralemmin-3.

    PubMed

    Chen, Xuxin; Wu, Xueling; Zhao, Yunfeng; Wang, Guansong; Feng, Jian; Li, Qi; Qian, Guisheng

    2011-01-28

    Previous studies have shown that single immunoglobulin IL-1 receptor-related molecule (SIGIRR) is a negative regulator of Toll-Interleukin-1 receptor signaling. Nevertheless, the molecular mechanism of the negatively regulatory effect of SIGIRR remains unknown. Using a yeast two-hybrid screen, we identified paralemmin-3 (PALM3) as a novel binding protein of SIGIRR. This interaction of SIGIRR with PALM3 was confirmed by coimmunoprecipitation in mammalian cells. In addition, the PALM3 mRNA expression was upregulated by lipopolysaccharide (LPS)-stimulation in a human alveolar epithelial cell line (A549 cells). Furthermore, silencing PALM3 by RNA interference inhibited the release of inflammatory cytokines in A549 cells after LPS-stimulation. These results suggest that PALM3 may function as an adaptor in the LPS- Toll-like receptor 4 signaling and the interaction of SIGIRR with PALM3 may partly account for the mechanism of the negatively regulatory effect of SIGIRR. Copyright © 2010 Elsevier Inc. All rights reserved.

  6. RNA targeting by small molecule alkaloids: Studies on the binding of berberine and palmatine to polyribonucleotides and comparison to ethidium

    NASA Astrophysics Data System (ADS)

    Islam, Md. Maidul; Suresh Kumar, Gopinatha

    2008-03-01

    The binding affinity, energetics and conformational aspects of the interaction of isoquinoline alkaloids berberine and palmatine to four single stranded polyribonucleotides polyguanylic acid [poly(G)], polyinosinic acid [poly(I)], polycytidylic acid [poly(C)] and polyuridylic acid [poly(U)] were studied by absorption, fluorescence, isothermal titration calorimetry and circular dichroism spectroscopy and compared with ethidium. Berberine, palmatine and ethidium binds strongly with poly(G) and poly(I) with affinity in the order 10 5 M -1 while their binding to poly(C) and poly(U) were very weak or practically nil. The same conclusions have also emerged from isothermal titration calorimetric studies. The binding of all the three compounds to poly(C) and poly(I) was exothermic and favored by both negative enthalpy change and positive entropy change. Conformational change in the polymer associated with the binding was observed in poly(I) with all the three molecules and poly(U) with ethidium but not in poly(G) and poly(C) revealing differences in the orientation of the bound molecules in the hitherto different helical organization of these polymers. These fundamental results may be useful and serve as database for the development of futuristic RNA based small molecule therapeutics.

  7. The murine cytomegalovirus immunoevasin gp40 binds MHC class I molecules to retain them in the early secretory pathway.

    PubMed

    Janßen, Linda; Ramnarayan, Venkat Raman; Aboelmagd, Mohamed; Iliopoulou, Maro; Hein, Zeynep; Majoul, Irina; Fritzsche, Susanne; Halenius, Anne; Springer, Sebastian

    2016-01-01

    In the presence of the murine cytomegalovirus (mCMV) gp40 (m152) protein, murine major histocompatibility complex (MHC) class I molecules do not reach the cell surface but are retained in an early compartment of the secretory pathway. We find that gp40 does not impair the folding or high-affinity peptide binding of the class I molecules but binds to them, leading to their retention in the endoplasmic reticulum (ER), the ER-Golgi intermediate compartment (ERGIC) and the cis-Golgi, most likely by retrieval from the cis-Golgi to the ER. We identify a sequence in gp40 that is required for both its own retention in the early secretory pathway and for that of class I molecules.

  8. Dye-Binding Assays for Evaluation of the Effects of Small Molecule Inhibitors on Amyloid (Aβ) Self-Assembly

    PubMed Central

    2012-01-01

    Dye-binding assays, such as those utilizing Congo red and thioflavin T, are among the most widely used tools to probe the aggregation of amyloidogenic biomolecules and for the evaluation of small molecule inhibitors of amyloid aggregation and fibrillization. A number of recent reports have indicated that these dye-binding assays could be prone to false positive effects when assessing inhibitors’ potential toward Aβ peptides, species involved in Alzheimer’s disease. Specifically, this review focuses on the application of thioflavin T for determining the efficiency of small molecule inhibitors of Aβ aggregation and addresses potential reasons that might be associated with the false positive effects in an effort to increase reliability of dye-binding assays. PMID:23173064

  9. The α-Hemolysin nanopore transduction detector – single-molecule binding studies and immunological screening of antibodies and aptamers

    PubMed Central

    Winters-Hilt, Stephen

    2007-01-01

    Background Nanopore detection is based on observations of the ionic current threading a single, highly stable, nanometer-scale channel. The dimensions are such that small biomolecules and biopolymers (like DNA and peptides) can translocate or be captured in the channel. The identities of translocating or captured molecules can often be discerned, one from another, based on their channel blockade "signatures". There is a self-limiting aspect to a translocation-based detection mechanism: as the channel fits tighter around the translocating molecule the dynamic range of the ionic current signal is reduced. In this study, a lengthy, highly structure, high dynamic-range, molecular capture is sought as a key component of a transduction-based nanopore detection platform. Results A specialized role, or device augmentation, involving bifunctional molecules has been explored. The bifunctional molecule has one function to enter and blockade the channel in an information-rich self-modulating manner, while the other function is for binding (usually), located on a non-channel-captured portion of the molecule. Part of the bifunctional molecule is, thus, external to the channel and is free to bind or rigidly link to a larger molecule of interest. What results is an event transduction detector: molecular events are directly transduced into discernible changes in the stationary statistics of the bifunctional molecule's channel blockade. Several results are presented of nanopore-based event-transduction detection. Conclusion It may be possible to directly track the bound versus unbound state of a huge variety of molecules using nanopore transduction detection. PMID:18047732

  10. Interactions of DNA binding proteins with G-Quadruplex structures at the single molecule level

    NASA Astrophysics Data System (ADS)

    Ray, Sujay

    Guanine-rich nucleic acid (DNA/RNA) sequences can form non-canonical secondary structures, known as G-quadruplex (GQ). Numerous in vivo and in vitro studies have demonstrated formation of these structures in telomeric and non-telomeric regions of the genome. Telomeric GQs protect the chromosome ends whereas non-telomeric GQs either act as road blocks or recognition sites for DNA metabolic machinery. These observations suggest the significance of these structures in regulation of different metabolic processes, such as replication and repair. GQs are typically thermodynamically more stable than the corresponding Watson-Crick base pairing formed by G-rich and C-rich strands, making protein activity a crucial factor for their destabilization. Inside the cell, GQs interact with different proteins and their enzymatic activity is the determining factor for their stability. We studied interactions of several proteins with GQs to understand the underlying principles of protein-GQ interactions using single-molecule FRET and other biophysical techniques. Replication Protein-A (RPA), a single stranded DNA (ssDNA) binding protein, is known to posses GQ unfolding activity. First, we compared the thermal stability of three potentially GQ-forming DNA sequences (PQS) to their stability against RPA-mediated unfolding. One of these sequences is the human telomeric repeat and the other two, located in the promoter region of tyrosine hydroxylase gene, are highly heterogeneous sequences that better represent PQS in the genome. The thermal stability of these structures do not necessarily correlate with their stability against protein-mediated unfolding. We conclude that thermal stability is not necessarily an adequate criterion for predicting the physiological viability of GQ structures. To determine the critical structural factors that influence protein-GQ interactions we studied two groups of GQ structures that have systematically varying loop lengths and number of G-tetrad layers. We

  11. Fusion of gelonin and anti-insulin-like growth factor-1 receptor (IGF-1R) affibody for enhanced brain cancer therapy.

    PubMed

    Ham, Songhee; Min, Kyoung Ah; Yang, Jae Wook; Shin, Meong Cheol

    2017-09-12

    Owing to the extraordinary potency in inhibiting protein translation that could eventually lead to apoptosis of tumor cells, ribosome-inactivating proteins (RIPs) such as gelonin have been considered attractive drug candidates for cancer therapy. However, due to several critical obstacles (e.g., severe toxicity issues caused by a lack of selectivity in their mode of action and the low cytotoxicity via poor cellular uptake, etc.), clinical application of RIPs is yet far from being accomplished. To overcome these challenges, in the present study, we engineered gelonin fusion proteins with anti-insulin-like growth factor-1 receptor (IGF-1R) affibody ("IAFF") via the genetic recombinant method and the SpyCatcher/SpyTag-mediated conjugation method. To this end, recombinant gelonin-anti-IGF-1R affibody (rGel-IAFF), gelonin-SpyCatcher (Gel-SpyCatcher) and SpyTag-IAFF fusion proteins were produced from the E. coli expression system, and gelonin-IAFF conjugate was synthesized by mixing Gel-SpyCatcher and SpyTag-IAFF. After preparation of both rGel-IAFF and Gel-IAFF conjugate, their components' functionality was characterized in vitro. Our assay results confirmed that, while both Gel-IAFF and Gel-SpyCatcher retained equipotent N-glycosidase activity to that of gelonin, IAFF was able to selectively bind to IGF-1R overexpressed U87 MG brain cancer cells over low expression LNCaP cells. The results of cellular analyses showed that rGel-IAFF and Gel-IAFF conjugate both exhibited a greater cell uptake in the U87 MG cells than gelonin, but not in the LNCaP cells, yielding a significantly augmented cytotoxicity only in the U87 MG cells. Remarkably, rGel-IAFF and Gel-IAFF conjugate displayed 22- and 5.6-fold lower IC50 values (avg. IC50: 180 and 720 nM, respectively) than gelonin (avg. IC50: 4000 nM) in the U87 MG cells. Overall, the results of the present research demonstrated that fusion of gelonin with IAFF could provide an effective way to enhance the anti-tumor activity

  12. In Silico Investigation of the Neurotensin Receptor 1 Binding Site: Overlapping Binding Modes for Small Molecule Antagonists and the Endogenous Peptide Agonist.

    PubMed

    Lückmann, Michael; Holst, Birgitte; Schwartz, Thue W; Frimurer, Thomas M

    2016-01-01

    The neurotensin receptor 1 (NTSR1) belongs to the family of 7TM, G protein-coupled receptors, and is activated by the 13-amino-acid peptide neurotensin (NTS) that has been shown to play important roles in neurological disorders and the promotion of cancer cells. Recently, a high-resolution x-ray crystal structure of NTSR1 in complex with NTS8-13 has been determined, providing novel insights into peptide ligand recognition by 7TM receptors. SR48692, a potent and selective small molecule antagonist has previously been used extensively as a tool compound to study NTSR1 receptor signaling properties. To investigate the binding mode of SR48692 and other small molecule compounds to NTSR1, we applied an Automated Ligand-guided Backbone Ensemble Receptor Optimization protocol (ALiBERO), taking receptor flexibility and ligand knowledge into account. Structurally overlapping binding poses for SR48692 and NTS8-13 were observed, despite their distinct chemical nature and inverse pharmacological profiles. The optimized models showed significantly improved ligand recognition in a large-scale virtual screening assessment compared to the crystal structure. Our models provide new insights into small molecule ligand binding to NTSR1 and could facilitate the structure-based design of non-peptide ligands for the evaluation of the pharmacological potential of NTSR1 in neurological disorders and cancer. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Identification of the binding site in intercellular adhesion molecule 1 for its receptor, leukocyte function-associated antigen 1.

    PubMed Central

    Fisher, K L; Lu, J; Riddle, L; Kim, K J; Presta, L G; Bodary, S C

    1997-01-01

    Intercellular adhesion molecule 1 (ICAM-1, CD54) is a member of the Ig superfamily and is a counterreceptor for the beta 2 integrins: lymphocyte function-associated antigen 1 (LFA-1, CD11a/CD18), complement receptor 1 (MAC-1, CD11b/CD18), and p150,95 (CD11c/CD18). Binding of ICAM-1 to these receptors mediates leukocyte-adhesive functions in immune and inflammatory responses. In this report, we describe a cell-free assay using purified recombinant extracellular domains of LFA-1 and a dimeric immunoadhesin of ICAM-1. The binding of recombinant secreted LFA-1 to ICAM-1 is divalent cation dependent (Mg2+ and Mn2+ promote binding) and sensitive to inhibition by antibodies that block LFA-1-mediated cell adhesion, indicating that its conformation mimics that of LFA-1 on activated lymphocytes. We describe six novel anti-ICAM-1 monoclonal antibodies, two of which are function blocking. Thirty-five point mutants of the ICAM-1 immunoadhesin were generated and residues important for binding of monoclonal antibodies and purified LFA-1 were identified. Nineteen of these mutants bind recombinant LFA-1 equivalently to wild type. Sixteen mutants show a 66-2500-fold decrease in LFA-1 binding yet, with few exceptions, retain binding to the monoclonal antibodies. These mutants, along with modeling studies, define the LFA-1 binding site on ICAM-1 as residues E34, K39, M64, Y66, N68, and Q73, that are predicted to lie on the CDFG beta-sheet of the Ig fold. The mutant G32A also abrogates binding to LFA-1 while retaining binding to all of the antibodies, possibly indicating a direct interaction of this residue with LFA-1. These data have allowed the generation of a highly refined model of the LFA-1 binding site of ICAM-1. Images PMID:9188101

  14. Screening of the Binding of Small Molecules to Proteins by Desorption Electrospray Ionization Mass Spectrometry Combined with Protein Microarray

    NASA Astrophysics Data System (ADS)

    Yao, Chenxi; Wang, Tao; Zhang, Buqing; He, Dacheng; Na, Na; Ouyang, Jin

    2015-11-01

    The interaction between bioactive small molecule ligands and proteins is one of the important research areas in proteomics. Herein, a simple and rapid method is established to screen small ligands that bind to proteins. We designed an agarose slide to immobilize different proteins. The protein microarrays were allowed to interact with different small ligands, and after washing, the microarrays were screened by desorption electrospray ionization mass spectrometry (DESI MS). This method can be applied to screen specific protein binding ligands and was shown for seven proteins and 34 known ligands for these proteins. In addition, a high-throughput screening was achieved, with the analysis requiring approximately 4 s for one sample spot. We then applied this method to determine the binding between the important protein matrix metalloproteinase-9 (MMP-9) and 88 small compounds. The molecular docking results confirmed the MS results, demonstrating that this method is suitable for the rapid and accurate screening of ligands binding to proteins.

  15. Thrombospondin-1 up-regulates expression of cell adhesion molecules and promotes monocyte binding to endothelium

    PubMed Central

    Narizhneva, Natalya V.; Razorenova, Olga V.; Podrez, Eugene A.; Chen, Juhua; Chandrasekharan, Unni M.; DiCorleto, Paul E.; Plow, Edward F.; Topol, Eric J.; Byzova, Tatiana V.

    2006-01-01

    Expression of cell adhesion molecules (CAM) responsible for leukocyte-endothelium interactions plays a crucial role in inflammation and atherogenesis. Up-regulation of vascular CAM-1 (VCAM-1), intracellular CAM-1 (ICAM-1), and E-selectin expression promotes monocyte recruitment to sites of injury and is considered to be a critical step in atherosclerotic plaque development. Factors that trigger this initial response are not well understood. As platelet activation not only promotes thrombosis but also early stages of atherogenesis, we considered the role of thrombospondin-1 (TSP-1), a matricellular protein released in abundance from activated platelets and accumulated in sites of vascular injury, as a regulator of CAM expression. TSP-1 induced expression of VCAM-1 and ICAM-1 on endothelium of various origins, which in turn, resulted in a significant increase of monocyte attachment. This effect could be mimicked by a peptide derived from the C-terminal domain of TSP-1 and known to interact with CD47 on the cell surface. The essential role of CD47 in the cellular responses to TSP-1 was demonstrated further using inhibitory antibodies and knockdown of CD47 with small interfering RNA. Furthermore, we demonstrated that secretion of endogenous TSP-1 and its interaction with CD47 on the cell surface mediates endothelial response to the major proinflammatory agent, tumor necrosis factor α (TNF-α). Taken together, this study identifies a novel mechanism regulating CAM expression and subsequent monocyte binding to endothelium, which might influence the development of anti-atherosclerosis therapeutic strategies. PMID:15833768

  16. Characterization of Small-Molecule Scaffolds That Bind to the Shigella Type III Secretion System Protein IpaD.

    PubMed

    Dey, Supratim; Anbanandam, Asokan; Mumford, Ben E; De Guzman, Roberto N

    2017-09-21

    Many pathogens such as Shigella and other bacteria assemble the type III secretion system (T3SS) nanoinjector to inject virulence proteins into their target cells to cause infectious diseases in humans. The rise of drug resistance among pathogens that rely on the T3SS for infectivity, plus the dearth of new antibiotics require alternative strategies in developing new antibiotics. The Shigella T3SS tip protein IpaD is an attractive target for developing anti-infectives because of its essential role in virulence and its exposure on the bacterial surface. Currently, the only known small molecules that bind to IpaD are bile salt sterols. In this study we identified four new small-molecule scaffolds that bind to IpaD, based on the methylquinoline, pyrrolidine-aniline, hydroxyindole, and morpholinoaniline scaffolds. NMR mapping revealed potential hotspots in IpaD for binding small molecules. These scaffolds can be used as building blocks in developing small-molecule inhibitors of IpaD that could lead to new anti-infectives. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Biochemical characterization of the selenoproteome in Gallus gallus via bioinformatics analysis: structure-function relationships and interactions of binding molecules.

    PubMed

    Zhu, Shi-Yong; Li, Xue-Nan; Sun, Xiao-Chen; Lin, Jia; Li, Wei; Zhang, Cong; Li, Jin-Long

    2017-02-22

    Knowledge about mammalian selenoproteins is increasing. However, the selenoproteome of birds remains considerably less understood, especially concerning its biochemical characterization, structure-function relationships and the interactions of binding molecules. In this work, the SECIS elements, subcellular localization, protein domains and interactions of binding molecules of the selenoproteome in Gallus gallus were analyzed using bioinformatics tools. We carried out comprehensive analyses of the structure-function relationships and interactions of the binding molecules of selenoproteins, to provide biochemical characterization of the selenoproteome in Gallus gallus. Our data provided a wealth of information on the biochemical functions of bird selenoproteins. Members of the selenoproteome were found to be involved in various biological processes in chickens, such as in antioxidants, maintenance of the redox balance, Se transport, and interactions with metals. Six membrane-bound selenoproteins (SelI, SelK, SelS, SelT, DIO1 and DIO3) played important roles in maintaining the membrane integrity. Chicken selenoproteins were classified according to their ligand binding sites as zinc-containing matrix metalloselenoproteins (Sep15, MsrB1, SelW and SelM), POP-containing selenoproteins (GPx1-4), FAD-interacting selenoproteins (TrxR1-3), secretory transport selenoproteins (GPx3 and SelPa) and other selenoproteins. The results of our study provided new evidence for the unknown biological functions of the selenoproteome in birds. Future research is required to confirm the novel biochemical functions of bird selenoproteins.

  18. Controllable binding of polar molecules and metastability of one-dimensional gases with attractive dipole forces.

    PubMed

    Byrd, Jason N; Montgomery, John A; Côté, Robin

    2012-08-24

    We explore one-dimensional samples of ultracold polar molecules with attractive dipole-dipole interactions and show the existence of a repulsive barrier caused by a strong quadrupole interaction between molecules. This barrier can stabilize a gas of ultracold KRb molecules and even lead to long-range wells supporting bound states between the molecules. The properties of these wells can be controlled by external electric fields, allowing the formation of long polymerlike chains of KRb and studies of quantum phase transitions by varying the effective interaction between molecules. We discuss the generalization of those results to other systems.

  19. A modern approach for epitope prediction: identification of foot-and-mouth disease virus peptides binding bovine leukocyte antigen (BoLA) class I molecules

    USDA-ARS?s Scientific Manuscript database

    Major histocompatibility complex (MHC) class I molecules regulate adaptive immune responses through the presentation of antigenic peptides to CD8positive T-cells. Polymorphisms in the peptide binding region of class I molecules determine peptide binding affinity and stability during antigen presenta...

  20. Identification of Small Molecule-binding Proteins in a Native Cellular Environment by Live-cell Photoaffinity Labeling.

    PubMed

    Head, Sarah A; Liu, Jun O

    2016-09-20

    Identifying the molecular target(s) of small molecules is a challenging but necessary step towards understanding their mechanism of action. While several target identification methods have been developed and used to successfully elucidate the binding proteins of a variety of small molecules, these techniques have drawbacks that make them unsuitable for detecting certain types of small molecule-target interactions. In particular, non-covalent interactions that depend on native cellular conditions, such as those of membrane proteins whose structures may be perturbed upon cell lysis, are often not amenable to affinity-based target identification methods. Here, we demonstrate a method wherein a probe containing a photolabile group is used to covalently crosslink to the small molecule binding protein within the environment of the live cell, allowing the detection and isolation of the target protein without the need for maintenance of the interaction after cell lysis. This technique is a valuable tool for studying biologically interesting small molecules with unknown mechanisms, both in the context of basic biology as well as drug discovery.

  1. Ligand-induced changes in 2-aminopurine fluorescence as a probe for small molecule binding to HIV-1 TAR RNA

    PubMed Central

    BRADRICK, THOMAS D.; MARINO, JOHN P.

    2004-01-01

    Replication of human immunodeficiency virus type 1 (HIV-1) is regulated in part through an interaction between the virally encoded trans-activator protein Tat and the trans-activator responsive region (TAR) of the viral RNA genome. Because TAR is highly conserved and its interaction with Tat is required for efficient viral replication, it has received much attention as an antiviral drug target. Here, we report a 2-aminopurine (2-AP) fluorescence-based assay for evaluating potential TAR inhibitors. Through selective incorporation of 2-AP within the bulge (C23 or U24) of a truncated form of the TAR sequence (Δ TAR-ap23 and Δ TAR-ap24), binding of argininamide, a 24-residue arginine-rich peptide derived from Tat, and Neomycin has been characterized using steady-state fluorescence. Binding of argininamide to the 2-AP ΔTAR constructs results in a four- to 11-fold increase in fluorescence intensity, thus providing a sensitive reporter of that interaction (KD ~ 1 mM). Similarly, binding of the Tat peptide results in an initial 14-fold increase in fluorescence (KD ~ 25 nM), but is then followed by a slight decrease that is attributed to an additional, lower-affinity association(s). Using the ΔTAR-ap23 and TAR-ap24 constructs, two classes of Neomycin binding sites are detected; the first molecule of antibiotic binds as a noncompetitive inhibitor of Tat/argininamide (KD ~ 200 nM), whereas the second, more weakly bound molecule(s) becomes associated in a presumably nonspecific manner (KD ~ 4 μM). Taken together, the results demonstrate that the 2-AP fluorescence-detected binding assays provide accurate and general methods for quantitatively assessing TAR interactions. PMID:15273324

  2. Rapid dynamics of general transcription factor TFIIB binding during preinitiation complex assembly revealed by single-molecule analysis

    PubMed Central

    Zhang, Zhengjian; English, Brian P.; Grimm, Jonathan B.; Kazane, Stephanie A.; Hu, Wenxin; Tsai, Albert; Inouye, Carla; You, Changjiang; Piehler, Jacob; Schultz, Peter G.; Lavis, Luke D.; Revyakin, Andrey; Tjian, Robert

    2016-01-01

    Transcription of protein-encoding genes in eukaryotic cells requires the coordinated action of multiple general transcription factors (GTFs) and RNA polymerase II (Pol II). A “step-wise” preinitiation complex (PIC) assembly model has been suggested based on conventional ensemble biochemical measurements, in which protein factors bind stably to the promoter DNA sequentially to build a functional PIC. However, recent dynamic measurements in live cells suggest that transcription factors mostly interact with chromatin DNA rather transiently. To gain a clearer dynamic picture of PIC assembly, we established an integrated in vitro single-molecule transcription platform reconstituted from highly purified human transcription factors and complemented it by live-cell imaging. Here we performed real-time measurements of the hierarchal promoter-specific binding of TFIID, TFIIA, and TFIIB. Surprisingly, we found that while promoter binding of TFIID and TFIIA is stable, promoter binding by TFIIB is highly transient and dynamic (with an average residence time of 1.5 sec). Stable TFIIB–promoter association and progression beyond this apparent PIC assembly checkpoint control occurs only in the presence of Pol II–TFIIF. This transient-to-stable transition of TFIIB-binding dynamics has gone undetected previously and underscores the advantages of single-molecule assays for revealing the dynamic nature of complex biological reactions. PMID:27798851

  3. Kinetic and equilibrium binding characterization of aptamers to small molecules using a label-free, sensitive, and scalable platform.

    PubMed

    Chang, Andrew L; McKeague, Maureen; Liang, Joe C; Smolke, Christina D

    2014-04-01

    Nucleic acid aptamers function as versatile sensing and targeting agents for analytical, diagnostic, therapeutic, and gene-regulatory applications, but their limited characterization and functional validation have hindered their broader implementation. We report the development of a surface plasmon resonance-based platform for rapid characterization of kinetic and equilibrium binding properties of aptamers to small molecules. Our system is label-free and scalable and enables analysis of different aptamer-target pairs and binding conditions with the same platform. This method demonstrates improved sensitivity, flexibility, and stability compared to other aptamer characterization methods. We validated our assay against previously reported aptamer affinity and kinetic measurements and further characterized a diverse panel of 12 small molecule-binding RNA and DNA aptamers. We report the first kinetic characterization for six of these aptamers and affinity characterization of two others. This work is the first example of direct comparison of in vitro selected and natural aptamers using consistent characterization conditions, thus providing insight into the influence of environmental conditions on aptamer binding kinetics and affinities, indicating different possible regulatory strategies used by natural aptamers, and identifying potential in vitro selection strategies to improve resulting binding affinities.

  4. MHC class I molecules are an essential cell surface component involved in Theileria parva sporozoite binding to bovine lymphocytes.

    PubMed

    Shaw, M K; Tilney, L G; Musoke, A J; Teale, A J

    1995-04-01

    The major histocompatibility complex (MHC) class I molecules are ubiquitous cell surface molecules involved in the cell-mediated immune response. We show here, using a number of different, independent approaches, that these proteins are an essential component of the host cell surface receptor involved in Theileria parva sporozoite invasion. Monoclonal antibodies (mAbs) reactive with common determinants on MHC class I molecules and with beta-2 microglobulin inhibited sporozoite entry by specifically preventing the initial binding event. However, in experiments using lymphocytes from heterozygous cattle in which at least four MHC class I gene products are expressed, mAbs which reacted with only one of these products did not inhibit entry. Using a series of bovine deletion mutant cell lines from which one or both MHC class I haplotypes had been lost, sporozoite binding and entry clearly correlated with the level of class I surface expression. While the level of sporozoite entry into cells in which one of the MHC class I haplotypes was lost was only slightly lower than into the parent cells, in a double deletion cell line having less than 5% of the class I expression of the parent cells the level of infection was only 4.3% of that into the parent cells. Furthermore, sporozoite entry into cells from a spontaneously arising mutant cell line exhibiting low levels of class I expression was correspondingly low. Treatment of lymphocytes with IL-2 produced a significant increase in host cell susceptibility and sporozoite entry and this increase correlated with either an increase in the number of target molecules per host cell, or in the binding of bovine MHC class I molecules to the mAbs. In particular, a significant increase in the level of reactivity with mAb W6/32 was observed. Lastly, we show that parasite entry can be competitively inhibited with an isolated sporozoite surface protein, p67. However, p67 binds weakly to lymphocyte surface molecules and initial attempts to

  5. Evidence of widespread binding of HLA class I molecules to peptides

    PubMed Central

    1990-01-01

    We have tested the binding of HLA class I proteins to peptides using a solid-phase binding assay. We tested 102 peptides, mostly derived from the HIV gag and HIV pol sequences. Most peptides did not bind to any class I protein tested. The pattern of binding among the three class I proteins tested, HLA-A2, -B27, and -B8, was approximately 85% concordant. Further, all five of the known HIV-1 gag T cell epitopes detected by human CTL bound at least one class I protein. Binding of class I to the peptides could be detected either by directly iodinated class I proteins, or indirectly using monoclonal antibodies specific for class I. The binding to the plates could be blocked with MA2.1, which binds in the alpha 1 region of A2, but not by W6/32, which binds elsewhere. The data presented here show that binding of class I to peptides is specific, but that many peptides bind to more than a single class I protein. PMID:2201749

  6. Respiratory complexes III and IV can each bind two molecules of cytochrome c at low ionic strength.

    PubMed

    Moreno-Beltrán, Blas; Díaz-Moreno, Irene; González-Arzola, Katiuska; Guerra-Castellano, Alejandra; Velázquez-Campoy, Adrián; De la Rosa, Miguel A; Díaz-Quintana, Antonio

    2015-02-13

    The transient interactions of respiratory cytochrome c with complexes III and IV is herein investigated by using heterologous proteins, namely human cytochrome c, the soluble domain of plant cytochrome c1 and bovine cytochrome c oxidase. The binding molecular mechanisms of the resulting cross-complexes have been analyzed by Nuclear Magnetic Resonance and Isothermal Titration Calorimetry. Our data reveal that the two cytochrome c-involving adducts possess a 2:1 stoichiometry - that is, two cytochrome c molecules per adduct - at low ionic strength. We conclude that such extra binding sites at the surfaces of complexes III and IV can facilitate the turnover and sliding of cytochrome c molecules and, therefore, the electron transfer within respiratory supercomplexes.

  7. Mapping protein pockets through their potential small-molecule binding volumes: QSCD applied to biological protein structures

    NASA Astrophysics Data System (ADS)

    Mason, Keith; Patel, Nehal M.; Ledel, Aric; Moallemi, Ciamac C.; Wintner, Edward A.

    2004-01-01

    Previously we demonstrated a method, Quantized Surface Complementarity Diversity (QSCD), of defining molecular diversity by measuring shape and functional complementarity of molecules to a basis set of theoretical target surfaces [Wintner E.A. and Moallemi C.C., J. Med. Chem., 43 (2000) 1993]. In this paper we demonstrate a method of mapping actual protein pockets to the same basis set of theoretical target surfaces, thereby allowing categorization of protein pockets by their properties of shape and functionality. The key step in the mapping is a `dissection' algorithm that breaks any protein pocket into a set of potential small molecule binding volumes. It is these binding volumes that are mapped to the basis set of theoretical target surfaces, thus measuring a protein pocket not as a single surface but as a collection of molecular recognition environments.

  8. Snyder-Robinson Syndrome: Rescuing the Disease-Causing Effect of G56S mutant by Small Molecule Binding

    NASA Astrophysics Data System (ADS)

    Zhang, Zhe; Martiny, Virginie; Lagorce, David; Alexov, Emil; Miteva, Maria; Clemson University Team; Université Paris Diderot Team

    2013-03-01

    Snyder-Robinson Syndrome (SRS) is an X-linked mental retardation disorder, which is caused by defects in a particular gene coding for the spermine synthase (SMS) protein. Among the missense mutations known to be disease-causing is the G56S, which is positioned at the interface of the SMS homo-dimer. Previous computational and experimental investigations have shown that G56S mutation destabilizes the homo-dimer and thus greatly reduces the SMS enzymatic activity. In this study, we explore the possibility of mitigating the effect of G56S mutation by binding small molecules to suitable pockets around the mutation site. It is done by combined efforts of molecular dynamics simulations and in silico screening. The binding of selected molecules was calculated to fully compensate the effect of the mutation and rescue the wild type dimer affinity. This work was supported by NIH, NLM grant. No. 1R03LM009748

  9. Small Molecule Binding, Docking, and Characterization of the Interaction between Pth1 and Peptidyl-tRNA

    SciTech Connect

    Hames, Mary C; McFeeters, Hana; Holloway, W Blake; Stanley, Christopher B; Urban, Volker S; McFeeters, Robert L

    2013-01-01

    Bacterial Pth1 is essential for viability. Pth1 cleaves the ester bond between the peptide and nucleotide of peptidyl-tRNA generated from aborted translation, expression of mini-genes, and short ORFs. We have determined the shape of the Pth1:peptidyl-tRNA complex using small angle neutron scattering. Binding of piperonylpiperazine, a small molecule constituent of a combinatorial synthetic library common to most compounds with inhibitory activity, was mapped to Pth1 via NMR spectroscopy. We also report computational docking results, modeling piperonylpiperazine binding based on chemical shift perturbation mapping. Overall these studies promote Pth1 as a novel antibiotic target, contributing to understanding how Pth1 interacts with its substrate, advancing the current model for cleavage, and demonstrating feasibility of small molecule inhibition.

  10. DNA Binding Peptide Directed Synthesis of Continuous DNA Nanowires for Analysis of Large DNA Molecules by Scanning Electron Microscope.

    PubMed

    Kim, Kyung-Il; Lee, Seonghyun; Jin, Xuelin; Kim, Su Ji; Jo, Kyubong; Lee, Jung Heon

    2017-01-01

    Synthesis of smooth and continuous DNA nanowires, preserving the original structure of native DNA, and allowing its analysis by scanning electron microscope (SEM), is demonstrated. Gold nanoparticles densely assembled on the DNA backbone via thiol-tagged DNA binding peptides work as seeds for metallization of DNA. This method allows whole analysis of DNA molecules with entangled 3D features. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Binding.

    ERIC Educational Resources Information Center

    Rebsamen, Werner

    1981-01-01

    Categorizes contemporary methods of binding printed materials in terms of physical preservation--hand binding (archival restoration), edition binding (paperback, hardcover), publication binding (magazines), textbook binding (sidesewn), single-sheet binding (loose-leaf, mechanical), and library binding (oversewn, sidesewn). Seven references are…

  12. Promiscuous binding of invariant chain-derived CLIP peptide to distinct HLA-I molecules revealed in leukemic cells.

    PubMed

    van Luijn, Marvin M; van de Loosdrecht, Arjan A; Lampen, Margit H; van Veelen, Peter A; Zevenbergen, Adri; Kester, Michel G D; de Ru, Arnoud H; Ossenkoppele, Gert J; van Hall, Thorbald; van Ham, S Marieke

    2012-01-01

    Antigen presentation by HLA class I (HLA-I) and HLA class II (HLA-II) complexes is achieved by proteins that are specific for their respective processing pathway. The invariant chain (Ii)-derived peptide CLIP is required for HLA-II-mediated antigen presentation by stabilizing HLA-II molecules before antigen loading through transient and promiscuous binding to different HLA-II peptide grooves. Here, we demonstrate alternative binding of CLIP to surface HLA-I molecules on leukemic cells. In HLA-II-negative AML cells, we found plasma membrane display of the CLIP peptide. Silencing Ii in AML cells resulted in reduced HLA-I cell surface display, which indicated a direct role of CLIP in the HLA-I antigen presentation pathway. In HLA-I-specific peptide eluates from B-LCLs, five Ii-derived peptides were identified, of which two were from the CLIP region. In vitro peptide binding assays strikingly revealed that the eluted CLIP peptide RMATPLLMQALPM efficiently bound to four distinct HLA-I supertypes (-A2, -B7, -A3, -B40). Furthermore, shorter length variants of this CLIP peptide also bound to these four supertypes, although in silico algorithms only predicted binding to HLA-A2 or -B7. Immunization of HLA-A2 transgenic mice with these peptides did not induce CTL responses. Together these data show a remarkable promiscuity of CLIP for binding to a wide variety of HLA-I molecules. The found participation of CLIP in the HLA-I antigen presentation pathway could reflect an aberrant mechanism in leukemic cells, but might also lead to elucidation of novel processing pathways or immune escape mechanisms.

  13. Measurement of the binding energy of ultracold 87Rb133Cs molecules using an offset-free optical frequency comb

    NASA Astrophysics Data System (ADS)

    Molony, Peter K.; Kumar, Avinash; Gregory, Philip D.; Kliese, Russell; Puppe, Thomas; Le Sueur, C. Ruth; Aldegunde, Jesus; Hutson, Jeremy M.; Cornish, Simon L.

    2016-08-01

    We report the binding energy of 87Rb133Cs molecules in their rovibrational ground state measured using an offset-free optical frequency comb based on difference frequency generation technology. We create molecules in the absolute ground state using stimulated Raman adiabatic passage (STIRAP) with a transfer efficiency of 88%. By measuring the absolute frequencies of our STIRAP lasers, we find the energy-level difference from an initial weakly bound Feshbach state to the rovibrational ground state with a resolution of ˜5 kHz over an energy-level difference of more than 114 T Hz ; this lets us discern the hyperfine splitting of the ground state. Combined with theoretical models of the Feshbach-state binding energies and ground-state hyperfine structure, we determine a zero-field binding energy of h ×114 268 135.24 (4 )(3 )M Hz . To our knowledge, this is the most accurate determination to date of the dissociation energy of a molecule.

  14. Using the binding site to control the magnetic and spintronic properties of a single magnetic molecule in a tunnel junction

    NASA Astrophysics Data System (ADS)

    Warner, Ben; El Hallak, Fadi; Prueser, Henning; Gill, Tobias G.; Sharp, John; Fisher, Andrew J.; Persson, Mats; Hirjibehedin, Cyrus F.

    2015-03-01

    Many proposals outline the use of single magnetic molecules in new applications in information technology and spintronics, with the intention of creating new devices based on phenomena that only manifest at the atomic scale. To create these devices it will be necessary to engineer the required properties, whether through controlling the molecule's chemical makeup or its interaction with the external surroundings. The latter may involve using interactions with the supporting substrate surface, which have been shown to not only modify the molecule properties but also create effects such as chirality. Here we utilize the surface interaction to modify the properties of FePc on copper nitride, a thin insulator, above bulk Cu(001). Using scanning tunneling microscopy we show that the interaction with the surface is defined by the binding site of the central Fe atom in the molecule. By performing elastic and inelastic tunneling spectroscopy and comparing the results to DFT modeling, we explore how coupling to the surface can be used to control the molecular orbitals and the accessibility of the spin excitations. This demonstrates the importance of controlling molecule-substrate coupling down to the atomic scale for the development of single molecule devices.

  15. Comparison of the binding of the therapeutically active nucleosides to DNA molecules with different level of lesions

    NASA Astrophysics Data System (ADS)

    Kruglova, E. B.; Gladkovskaya, N. A.

    2002-12-01

    Recently we have shown that DNA molecules extracted from epididymis of the Wistar male rats exposed to low doses of gamma radiation interact with some pyrimidine nucleosides. The bindign affinities of NUC to control DNA molecules are unessential. Comparing the UV melting curves for the various DNA sammples we show that observed differences are related to conformational chagnes in the DNA double helix. The samples of the damaged DNA have been obtained by partial denaturation of the calf thymus DNA in the salt-free aqueous solutions. The level of DNA damages in the model DNA smplase depends on the DNA concentration. It was shown that damages in the DNA molecules lead to changes of the melting curves of DNA-NUC mixtures that are similar to those for the DNA samples extracted from irradiated tissues. ALso it has been found that the binding mechanisms to cytosine arabinoside and 6-azacytosine to DNA molecuels having modifeid secondary structures are different.

  16. Screening Mixtures of Small Molecules for Binding to Multiple Sites on the Surface Tetanus Toxin C Fragment by Bioaffinity NMR

    SciTech Connect

    Cosman, M; Zeller, L; Lightstone, F C; Krishnan, V V; Balhorn, R

    2002-01-01

    The clostridial neurotoxins include the closely related tetanus (TeNT) and botulinum (BoNT) toxins. Botulinum toxin is used to treat severe muscle disorders and as a cosmetic wrinkle reducer. Large quantities of botulinum toxin have also been produced by terrorists for use as a biological weapon. Because there are no known antidotes for these toxins, they thus pose a potential threat to human health whether by an accidental overdose or by a hostile deployment. Thus, the discovery of high specificity and affinity compounds that can inhibit their binding to neural cells can be used as antidotes or in the design of chemical detectors. Using the crystal structure of the C fragment of the tetanus toxin (TetC), which is the cell recognition and cell surface binding domain, and the computational program DOCK, sets of small molecules have been predicted to bind to two different sites located on the surface of this protein. While Site-1 is common to the TeNT and BoNTs, Site-2 is unique to TeNT. Pairs of these molecules from each site can then be linked together synthetically to thereby increase the specificity and affinity for this toxin. Electrospray ionization mass spectroscopy was used to experimentally screen each compound for binding. Mixtures containing binders were further screened for activity under biologically relevant conditions using nuclear magnetic resonance (NMR) methods. The screening of mixtures of compounds offers increased efficiency and throughput as compared to testing single compounds and can also evaluate how possible structural changes induced by the binding of one ligand can influence the binding of the second ligand. In addition, competitive binding experiments with mixtures containing ligands predicted to bind the same site could identify the best binder for that site. NMR transfer nuclear Overhauser effect (trNOE) confirm that TetC binds doxorubicin but that this molecule is displaced by N-acetylneuraminic acid (sialic acid) in a mixture that

  17. Affilin-novel binding molecules based on human gamma-B-crystallin, an all beta-sheet protein.

    PubMed

    Ebersbach, Hilmar; Fiedler, Erik; Scheuermann, Tanja; Fiedler, Markus; Stubbs, Milton T; Reimann, Carola; Proetzel, Gabriele; Rudolph, Rainer; Fiedler, Ulrike

    2007-09-07

    The concept of novel binding proteins as an alternative to antibodies has undergone rapid development and is now ready for practical use in a wide range of applications. Alternative binding proteins, based on suitable scaffolds with desirable properties, are selected from combinatorial libraries in vitro. Here, we describe an approach using a beta-sheet of human gamma-B-crystallin to generate a universal binding site through randomization of eight solvent-exposed amino acid residues selected according to structural and sequence analyses. Specific variants, so-called Affilin, have been isolated from a phage display library against a variety of targets that differ considerably in size and structure. The isolated Affilin variants can be produced in Escherichia coli as soluble proteins and have a high level of thermodynamic stability. The crystal structures of the human wild-type gamma-B-crystallin and a selected Affilin variant have been determined to 1.7 A and 2.0 A resolution, respectively. Comparison of the two molecules indicates that the human gamma-B-crystallin tolerates amino acid exchanges with no major structural change. We conclude that the intrinsically stable and easily expressed gamma-B-crystallin provides a suitable framework for the generation of novel binding molecules.

  18. Limited Proteolysis Combined with Stable Isotope Labeling Reveals Conformational Changes in Protein (Pseudo)kinases upon Binding Small Molecules.

    PubMed

    Di Michele, Michela; Stes, Elisabeth; Vandermarliere, Elien; Arora, Rohit; Astorga-Wells, Juan; Vandenbussche, Jonathan; van Heerde, Erika; Zubarev, Roman; Bonnet, Pascal; Linders, Joannes T M; Jacoby, Edgar; Brehmer, Dirk; Martens, Lennart; Gevaert, Kris

    2015-10-02

    Likely due to conformational rearrangements, small molecule inhibitors may stabilize the active conformation of protein kinases and paradoxically promote tumorigenesis. We combined limited proteolysis with stable isotope labeling MS to monitor protein conformational changes upon binding of small molecules. Applying this method to the human serine/threonine kinase B-Raf, frequently mutated in cancer, we found that binding of ATP or its nonhydrolyzable analogue AMP-PNP, but not ADP, stabilized the structure of both B-Raf(WT) and B-Raf(V600E). The ATP-competitive type I B-Raf inhibitor vemurafenib and the type II inhibitor sorafenib stabilized the kinase domain (KD) but had distinct effects on the Ras-binding domain. Stabilization of the B-Raf(WT) KD was confirmed by hydrogen/deuterium exchange MS and molecular dynamics simulations. Our results are further supported by cellular assays in which we assessed cell viability and phosphorylation profiles in cells expressing B-Raf(WT) or B-Raf(V600E) in response to vemurafenib or sorafenib. Our data indicate that an overall stabilization of the B-Raf structure by specific inhibitors activates MAPK signaling and increases cell survival, helping to explain clinical treatment failure. We also applied our method to monitor conformational changes upon nucleotide binding of the pseudokinase KSR1, which holds high potential for inhibition in human diseases.

  19. Fundamentals of large-molecule protein therapeutic bioanalysis using ligand-binding assays.

    PubMed

    Thway, Theingi M

    2016-01-01

    This article provides an overview of ligand-binding assays, including the origin and evolution of the primary concepts, in addition to reviewing commonly used assay formats. The birth of ligand-binding assays began with a radioimmunoassay developed to measure insulin in 1960. Fundamental to ligand-binding assay design is the requirement of at least one protein that interacts with the analyte of interest. Enzyme immunoassay has largely supplanted radioimmunoassay as the ligand-binding assay of choice in today's laboratory environment. This article illustrates various assay formats such as competitive, sandwich and bridging, in addition to, describing critical reagents necessary for their design. The utility of ligand-binding assays in therapeutic protein development and comparison to alternative bioanalysis platforms is discussed.

  20. Identification of Grb2 as a novel binding partner of the signaling lymphocytic activation molecule-associated protein binding receptor CD229.

    PubMed

    Martín, Margarita; Del Valle, Juana M; Saborit, Ifigènia; Engel, Pablo

    2005-05-15

    Ag recognition by the TCR determines the subsequent fate of the T cell and is regulated by the involvement of other cell surface molecules, termed coreceptors. CD229 is a lymphocyte cell surface molecule that belongs to the CD150 family of receptors. Upon tyrosine phosphorylation, CD229 recruits various signaling molecules to the membrane. One of these molecules is the signaling lymphocytic activation molecule-associated protein, of which a deficiency leads to the X-linked lymphoproliferative syndrome. We report that CD229 interacts in a phosphorylation-dependent manner with Grb2. We mapped this interaction showing that the Src homology 2 domain of Grb2 and the tyrosine residue Y606 in CD229 are required for CD229-Grb2 complex formation. The Grb2 motif in the cytoplasmic tail of CD229 is distinct and independent from the two tyrosines required for efficient signaling lymphocytic activation molecule-associated protein recruitment. CD229, but not other members of the CD150 family, directly bound Grb2. We also demonstrate that CD229 precipitates with Grb2 in T lymphocytes after pervanadate treatment, as well as CD229 or TCR ligation. Interestingly, the CD229 mutant lacking the Grb2 binding site is not internalized after CD229 engagement with specific Abs. Moreover, a dominant negative form of Grb2 (containing only Src homology 2 domain) impaired CD229 endocytosis. Unexpectedly, Erk phosphorylation was partially inhibited after activation of CD229 plus CD3. Consistent with this, CD229 ligation partially inhibited TCR signaling in peripheral blood cells and CD229-Jurkat cells transfected with the 3XNFAT-luciferase reporter construct. Altogether, the data suggest a model whereby CD229 ligation attenuates TCR signaling and Grb2 recruitment to CD229 controls its rate of internalization.

  1. Quantifying the Entropy of Binding for Water Molecules in Protein Cavities by Computing Correlations

    PubMed Central

    Huggins, David J.

    2015-01-01

    Protein structural analysis demonstrates that water molecules are commonly found in the internal cavities of proteins. Analysis of experimental data on the entropies of inorganic crystals suggests that the entropic cost of transferring such a water molecule to a protein cavity will not typically be greater than 7.0 cal/mol/K per water molecule, corresponding to a contribution of approximately +2.0 kcal/mol to the free energy. In this study, we employ the statistical mechanical method of inhomogeneous fluid solvation theory to quantify the enthalpic and entropic contributions of individual water molecules in 19 protein cavities across five different proteins. We utilize information theory to develop a rigorous estimate of the total two-particle entropy, yielding a complete framework to calculate hydration free energies. We show that predictions from inhomogeneous fluid solvation theory are in excellent agreement with predictions from free energy perturbation (FEP) and that these predictions are consistent with experimental estimates. However, the results suggest that water molecules in protein cavities containing charged residues may be subject to entropy changes that contribute more than +2.0 kcal/mol to the free energy. In all cases, these unfavorable entropy changes are predicted to be dominated by highly favorable enthalpy changes. These findings are relevant to the study of bridging water molecules at protein-protein interfaces as well as in complexes with cognate ligands and small-molecule inhibitors. PMID:25692597

  2. Quantifying the entropy of binding for water molecules in protein cavities by computing correlations.

    PubMed

    Huggins, David J

    2015-02-17

    Protein structural analysis demonstrates that water molecules are commonly found in the internal cavities of proteins. Analysis of experimental data on the entropies of inorganic crystals suggests that the entropic cost of transferring such a water molecule to a protein cavity will not typically be greater than 7.0 cal/mol/K per water molecule, corresponding to a contribution of approximately +2.0 kcal/mol to the free energy. In this study, we employ the statistical mechanical method of inhomogeneous fluid solvation theory to quantify the enthalpic and entropic contributions of individual water molecules in 19 protein cavities across five different proteins. We utilize information theory to develop a rigorous estimate of the total two-particle entropy, yielding a complete framework to calculate hydration free energies. We show that predictions from inhomogeneous fluid solvation theory are in excellent agreement with predictions from free energy perturbation (FEP) and that these predictions are consistent with experimental estimates. However, the results suggest that water molecules in protein cavities containing charged residues may be subject to entropy changes that contribute more than +2.0 kcal/mol to the free energy. In all cases, these unfavorable entropy changes are predicted to be dominated by highly favorable enthalpy changes. These findings are relevant to the study of bridging water molecules at protein-protein interfaces as well as in complexes with cognate ligands and small-molecule inhibitors.

  3. Small-molecule inhibitor binding to an N-acyl-homoserine lactone synthase.

    PubMed

    Chung, Jiwoung; Goo, Eunhye; Yu, Sangheon; Choi, Okhee; Lee, Jeehyun; Kim, Jinwoo; Kim, Hongsup; Igarashi, Jun; Suga, Hiroaki; Moon, Jae Sun; Hwang, Ingyu; Rhee, Sangkee

    2011-07-19

    Quorum sensing (QS) controls certain behaviors of bacteria in response to population density. In gram-negative bacteria, QS is often mediated by N-acyl-L-homoserine lactones (acyl-HSLs). Because QS influences the virulence of many pathogenic bacteria, synthetic inhibitors of acyl-HSL synthases might be useful therapeutically for controlling pathogens. However, rational design of a potent QS antagonist has been thwarted by the lack of information concerning the binding interactions between acyl-HSL synthases and their ligands. In the gram-negative bacterium Burkholderia glumae, QS controls virulence, motility, and protein secretion and is mediated by the binding of N-octanoyl-L-HSL (C8-HSL) to its cognate receptor, TofR. C8-HSL is synthesized by the acyl-HSL synthase TofI. In this study, we characterized two previously unknown QS inhibitors identified in a focused library of acyl-HSL analogs. Our functional and X-ray crystal structure analyses show that the first inhibitor, J8-C8, binds to TofI, occupying the binding site for the acyl chain of the TofI cognate substrate, acylated acyl-carrier protein. Moreover, the reaction byproduct, 5'-methylthioadenosine, independently binds to the binding site for a second substrate, S-adenosyl-L-methionine. Closer inspection of the mode of J8-C8 binding to TofI provides a likely molecular basis for the various substrate specificities of acyl-HSL synthases. The second inhibitor, E9C-3oxoC6, competitively inhibits C8-HSL binding to TofR. Our analysis of the binding of an inhibitor and a reaction byproduct to an acyl-HSL synthase may facilitate the design of a new class of QS-inhibiting therapeutic agents.

  4. Five HLA-DP Molecules Frequently Expressed in the Worldwide Human Population Share a Common HLA Supertypic Binding Specificity

    PubMed Central

    Sidney, John; Steen, Amiyah; Moore, Carrie; Ngo, Sandy; Chung, Jolan; Peters, Bjoern; Sette, Alessandro

    2010-01-01

    Compared with DR and DQ, knowledge of the binding repertoires and specificities of HLA-DP alleles is somewhat limited. However, a growing body of literature has indicated the importance of DP-restricted responses in the context of cancer, allergy, and infectious disease. In the current study, we developed high-throughput binding assays for the five most common HLA-DPB1 alleles in the general worldwide population. Using these assays on a comprehensive panel of single-substitution analogs and large peptide libraries, we derived novel detailed binding motifs for DPB1*0101 and DPB1*0501. We also derived more detailed quantitative motifs for DPB1*0201, DPB1*0401, and DPB1*0402, which were previously characterized on the basis of sets of eluted ligands and/or limited sets of substituted peptides. Unexpectedly, all five DP molecules, originally selected only on the basis of their frequency in human populations, were found to share largely overlapping peptide motifs. Testing panels of known DP epitopes and a panel of peptides spanning a set of Phleum pratense Ags revealed that these molecules also share largely overlapping peptide-binding repertoires. This demonstrates that a previously hypothesized DP supertype extends far beyond what was originally envisioned and includes at least three additional very common DP specificities. Taken together, these DP supertype molecules are found in >90% of the human population. Thus, these findings have important implications for epitope-identification studies and monitoring of human class II-restricted immune responses. PMID:20139279

  5. Dehydroepiandrosterone (DHEA) Inhibition of Monocyte Binding by Vascular Endothelium Is Associated With Sialylation of Neural Cell Adhesion Molecule

    PubMed Central

    Curatola, Anna-Maria; Huang, Kui; Naftolin, Frederick

    2012-01-01

    Rationale: Adhesion of monocytes to vascular endothelium is necessary for atheroma formation. This adhesion requires binding of endothelial neural cell adhesion molecule (NCAM) to monocyte NCAM. NCAM:NCAM binding is blocked by sialylation of NCAM (polysialylated NCAM; PSA-NCAM). Since estradiol (E2) and dihydrotestosterone (DHT) induced PSA-NCAM and decreased monocyte adhesion, in consideration of possible clinical applications we tested whether their prohormone dehydroepiandrosterone (DHEA) has similar effects. Experimental: (1) DHEA was administered to cultured human coronary artery endothelial cells (HCAECs) from men and women. Monocyte binding was assessed using fluorescence-labeled monocytes. (2) HCEACs were incubated with E2, DHT, DHEA alone, or with trilostane, fulvestrant or flutamide. Expression of PSA-NCAM was assessed by immunohistochemistry and Western blotting. Results: Dehydroepiandrosterone inhibited monocyte adhesion to HCAECs by ≥50% (P < .01). Fulvestrant or flutamide blockade of DHEA’s inhibition of monocyte binding appeared to be gender dependent. The DHEA-induced expression of PSA-NCAM was completely blocked by trilostane. Conclusions: In these preliminary in vitro studies, DHEA increased PSA-NCAM expression and inhibited monocyte binding in an estrogen- and androgen receptor-dependent manner. Dehydroepiandrosteroneappears to act via its end metabolites, E2 and DHT. Dehydroepiandrosterone could furnish clinical prevention against atherogenesis and arteriosclerosis. PMID:22228741

  6. Dehydroepiandrosterone (DHEA) inhibition of monocyte binding by vascular endothelium is associated with sialylation of neural cell adhesion molecule.

    PubMed

    Curatola, Anna-Maria; Huang, Kui; Naftolin, Frederick

    2012-01-01

    Adhesion of monocytes to vascular endothelium is necessary for atheroma formation. This adhesion requires binding of endothelial neural cell adhesion molecule (NCAM) to monocyte NCAM. NCAM:NCAM binding is blocked by sialylation of NCAM (polysialylated NCAM; PSA-NCAM). Since estradiol (E2) and dihydrotestosterone (DHT) induced PSA-NCAM and decreased monocyte adhesion, in consideration of possible clinical applications we tested whether their prohormone dehydroepiandrosterone (DHEA) has similar effects. (1) DHEA was administered to cultured human coronary artery endothelial cells (HCAECs) from men and women. Monocyte binding was assessed using fluorescence-labeled monocytes. (2) HCEACs were incubated with E2, DHT, DHEA alone, or with trilostane, fulvestrant or flutamide. Expression of PSA-NCAM was assessed by immunohistochemistry and Western blotting. Dehydroepiandrosterone inhibited monocyte adhesion to HCAECs by ≥50% (P < .01). Fulvestrant or flutamide blockade of DHEA's inhibition of monocyte binding appeared to be gender dependent. The DHEA-induced expression of PSA-NCAM was completely blocked by trilostane. In these preliminary in vitro studies, DHEA increased PSA-NCAM expression and inhibited monocyte binding in an estrogen- and androgen receptor-dependent manner. Dehydroepiandrosteroneappears to act via its end metabolites, E2 and DHT. Dehydroepiandrosterone could furnish clinical prevention against atherogenesis and arteriosclerosis.

  7. Binding Energy of Molecules on Water Ice: Laboratory Measurements and Modeling

    NASA Astrophysics Data System (ADS)

    He, Jiao; Acharyya, Kinsuk; Vidali, Gianfranco

    2016-07-01

    We measured the binding energy of N2, CO, O2, CH4, and CO2 on non-porous (compact) amorphous solid water (np-ASW), of N2 and CO on porous ASW, and of NH3 on crystalline water ice. We were able to measure binding energies down to a fraction of 1% of a layer, thus making these measurements more appropriate for astrochemistry than the existing values. We found that CO2 forms clusters on the np-ASW surface even at very low coverages. The binding energies of N2, CO, O2, and CH4 decrease with coverage in the submonolayer regime. Their values at the low coverage limit are much higher than what is commonly used in gas-grain models. An empirical formula was used to describe the coverage dependence of the binding energies. We used the newly determined binding energy distributions in a simulation of gas-grain chemistry for cold cloud and hot-core models. We found that owing to the higher value of binding energy in the submonolayer regime, a fraction of all these ices remains for much longer and up to higher temperatures on the grain surface compared to the single value energies currently used in the astrochemical models.

  8. Basic fibroblast growth factor binds to subendothelial extracellular matrix and is released by heparitinase and heparin-like molecules

    SciTech Connect

    Bashkin, P.; Doctrow, S.; Klagsbrun, M.; Svahn, C.M.; Folkman, J.; Vlodavsky, I. )

    1989-02-21

    Basic fibroblast growth factor (bFGF) exhibits specific binding to the extracellular matrix (ECM) produced by cultured endothelial cells. Binding was saturable as a function both of time and of concentration of {sup 125}I-bFGF. Scatchard analysis of FGF binding revealed the presence of about 1.5 x 10{sup 12} binding sites/mm{sup 2} ECM with an apparent k{sub D} of 610 nM. FGF binds to heparan sulfate (HS) in ECM as evidenced by (i) inhibition of binding in the presence of heparin or HS at 0.1-1 {mu}g/mL, but not by chondroitin sulfate, keratan sulfate, or hyaluronic acid at 10 {mu}g/mL, (ii) lack of binding to ECM pretreated with heparitinase, but not with chondroitinase ABC, and (iii) rapid release of up to 90% of ECM-bound FGF by exposure to heparin, HS, or heparitinase, but not to chondroitin sulfate, keratan sulfate, hyaluronic acid, or chondroitinase ABC. Oligosaccharides derived from depolymerized heparin, and as small as the tetrasaccharide, released the ECM-bound FGF, but there was little or no release of FGF by modified nonanticoagulant heparins such as totally desulfated heparin, N-desulfated heparin, and N-acetylated heparin. FGF released from ECM was biologically active, as indicated by its stimulation of cell proliferation and DNA synthesis in vascular endothelial cells and 3T3 fibroblasts. Similar results were obtained in studies on release of endogenous FGF-like mitogenic activity from Descement's membranes of bovine corneas. It is suggested that ECM storage and release of bFGF provide a novel mechanism for regulation of capillary blood vessel growth. Whereas ECM-bound FGF may be prevented from acting on endothelial cells, its displacement by heparin-like molecules and/or HS-degrading enzymes may elicit a neovascular response.

  9. Ligand-mediated dimerization of a carbohydrate-binding molecule reveals a novel mechanism for protein-carbohydrate recognition.

    PubMed

    Flint, James; Nurizzo, Didier; Harding, Stephen E; Longman, Emma; Davies, Gideon J; Gilbert, Harry J; Bolam, David N

    2004-03-19

    The structural and thermodynamic basis for carbohydrate-protein recognition is of considerable importance. NCP-1, which is a component of the Piromyces equi cellulase/hemicellulase complex, presents a provocative model for analyzing how structural and mutational changes can influence the ligand specificity of carbohydrate-binding proteins. NCP-1 contains two "family 29" carbohydrate-binding modules designated CBM29-1 and CBM29-2, respectively, that display unusually broad specificity; the proteins interact weakly with xylan, exhibit moderate affinity for cellulose and mannan, and bind tightly to the beta-1,4-linked glucose-mannose heteropolymer glucomannan. The crystal structure of CBM29-2 in complex with cellohexaose and mannohexaose identified key residues involved in ligand recognition. By exploiting this structural information and the broad specificity of CBM29-2, we have used this protein as a template to explore the evolutionary mechanisms that can lead to significant changes in ligand specificity. Here, we report the properties of the E78R mutant of CBM29-2, which displays ligand specificity that is different from that of wild-type CBM29-2; the protein retains significant affinity for cellulose but does not bind to mannan or glucomannan. Significantly, E78R exhibits a stoichiometry of 0.5 when binding to cellohexaose, and both calorimetry and ultracentrifugation show that the mutant protein displays ligand-mediated dimerization in solution. The three-dimensional structure of E78R in complex with cellohexaose reveals the intriguing molecular basis for this "dimeric" binding mode that involves the lamination of the oligosaccharide between two CBM molecules. The 2-fold screw axis of the ligand is mirrored in the orientation of the two protein domains with adjacent sugar rings stacking against the equivalent aromatic residues in the binding site of each protein molecule of the molecular sandwich. The sandwiching of an oligosaccharide chain between two protein

  10. Fighting Cholera One-on-One: The Development and Efficacy of Multivalent Cholera-Toxin-Binding Molecules.

    PubMed

    Zuilhof, Han

    2016-02-16

    A series of diseases, ranging from cholera via travelers' diarrhea to hamburger disease, are caused by bacterially produced toxic proteins. In particular, a toxic protein unit is brought into the host cell upon binding to specific membrane-bound oligosaccharides on the host cell membrane. For example, the protein that causes cholera, cholera toxin (CT), has five identical, symmetrically placed binding pockets (B proteins), on top of which the toxic A protein resides. A promising strategy to counteract the devastating biological effects of this AB5 protein involves the development of inhibitors that can act as mimics of membrane-bound GM1 molecules, i.e., that can bind CT strongly and selectively. To reach this goal, two features are essential: First of all, the inhibitor should display oligosaccharides that resemble as much as possible the naturally occurring cell-surface pentasaccharide onto which CT normally binds, the so-called GM1 sugar (the oligosaccharide part of which is then labeled GM1os). Second, the inhibitor should be able to bind CT via multivalent interactions so as to bind CT as strongly as possible to allow for a real competition with the cell-membrane-bound GM1 molecules. In this Account, we present elements of the path that leads to strong CT inhibition by outlining the roles of multivalency and the development and use of GM1 mimics. First, multivalency effects were investigated using "sugar-coated" platforms, ranging from dendritic structures with up to eight oligosaccharides to platforms that mimicked the fivefold symmetry of CT itself. The latter goal was reached either via synthetic scaffolds like corannulene or calix[5]arene or via the development of a neolectin CT mimic that itself carries five GM1os groups. Second, the effect of the nature of the oligosaccharide appended to this platform was investigated via the use of oligosaccharides of increasing complexity, from galactose and lactose to the tetrasaccharide GM2os and eventually to GM1os

  11. Isolation of specific genomic regions and identification of associated molecules by engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) using CRISPR.

    PubMed

    Fujita, Toshitsugu; Fujii, Hodaka

    2015-01-01

    Isolation of specific genomic regions retaining molecular interactions is necessary for their biochemical analysis. Here, we describe engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) using the CRISPR system, for purification of specific genomic regions retaining molecular interactions. In this form of enChIP, specific genomic regions are immunoprecipitated with antibody against a tag(s), which is fused to a catalytically inactive form of Cas9 (dCas9), which is co-expressed with a guide RNA (gRNA) and recognizes endogenous DNA sequence in the genomic regions of interest. enChIP combined with mass spectrometry (enChIP-MS), next-generation sequencing (enChIP-Seq), and RNA-Seq (enChIP-RNA-Seq) can identify proteins, other genomic regions, and RNA, respectively, that interact with the target genomic region.

  12. Small molecule inhibition of cAMP response element binding protein in human acute myeloid leukemia cells.

    PubMed

    Mitton, B; Chae, H-D; Hsu, K; Dutta, R; Aldana-Masangkay, G; Ferrari, R; Davis, K; Tiu, B C; Kaul, A; Lacayo, N; Dahl, G; Xie, F; Li, B X; Breese, M R; Landaw, E M; Nolan, G; Pellegrini, M; Romanov, S; Xiao, X; Sakamoto, K M

    2016-12-01

    The transcription factor CREB (cAMP Response-Element Binding Protein) is overexpressed in the majority of acute myeloid leukemia (AML) patients, and this is associated with a worse prognosis. Previous work revealed that CREB overexpression augmented AML cell growth, while CREB knockdown disrupted key AML cell functions in vitro. In contrast, CREB knockdown had no effect on long-term hematopoietic stem cell activity in mouse transduction/transplantation assays. Together, these studies position CREB as a promising drug target for AML. To test this concept, a small molecule inhibitor of CREB, XX-650-23, was developed. This molecule blocks a critical interaction between CREB and its required co-activator CBP (CREB Binding Protein), leading to disruption of CREB-driven gene expression. Inhibition of CBP-CREB interaction induced apoptosis and cell-cycle arrest in AML cells, and prolonged survival in vivo in mice injected with human AML cells. XX-650-23 had little toxicity on normal human hematopoietic cells and tissues in mice. To understand the mechanism of XX-650-23, we performed RNA-seq, ChIP-seq and Cytometry Time of Flight with human AML cells. Our results demonstrate that small molecule inhibition of CBP-CREB interaction mostly affects apoptotic, cell-cycle and survival pathways, which may represent a novel approach for AML therapy.

  13. Mechanism of Collaborative Enhancement of Binding of Paired Antibodies to Distinct Epitopes of Platelet Endothelial Cell Adhesion Molecule-1

    PubMed Central

    Greineder, Colin F.; Villa, Carlos H.; Hood, Elizabeth D.; Shuvaev, Vladimir V.; Sun, Jing; Chacko, Ann-Marie; Abraham, Valsamma; DeLisser, Horace M.; Muzykantov, Vladimir R.

    2017-01-01

    Monoclonal antibodies (mAbs) directed to extracellular epitopes of human and mouse Platelet Endothelial Cell Adhesion Molecule-1 (CD31 or PECAM-1) stimulate binding of other mAbs to distinct adjacent PECAM-1 epitopes. This effect, dubbed Collaborative Enhancement of Paired Affinity Ligands, or CEPAL, has been shown to enhance delivery of mAb-targeted drugs and nanoparticles to the vascular endothelium. Here we report new insights into the mechanism underlying this effect, which demonstrates equivalent amplitude in the following models: i) cells expressing a full length PECAM-1 and mutant form of PECAM-1 unable to form homodimers; ii) isolated fractions of cellular membranes; and, iii) immobilized recombinant PECAM-1. These results indicate that CEPAL is mediated not by interference in cellular functions or homophilic PECAM-1 interactions, but rather by conformational changes within the cell adhesion molecule induced by ligand binding. This mechanism, mediated by exposure of partially occult epitopes, is likely to occur in molecules other than PECAM-1 and may represent a generalizable phenomenon with valuable practical applications. PMID:28085903

  14. Mechanism of Collaborative Enhancement of Binding of Paired Antibodies to Distinct Epitopes of Platelet Endothelial Cell Adhesion Molecule-1.

    PubMed

    Kiseleva, Raisa; Greineder, Colin F; Villa, Carlos H; Hood, Elizabeth D; Shuvaev, Vladimir V; Sun, Jing; Chacko, Ann-Marie; Abraham, Valsamma; DeLisser, Horace M; Muzykantov, Vladimir R

    2017-01-01

    Monoclonal antibodies (mAbs) directed to extracellular epitopes of human and mouse Platelet Endothelial Cell Adhesion Molecule-1 (CD31 or PECAM-1) stimulate binding of other mAbs to distinct adjacent PECAM-1 epitopes. This effect, dubbed Collaborative Enhancement of Paired Affinity Ligands, or CEPAL, has been shown to enhance delivery of mAb-targeted drugs and nanoparticles to the vascular endothelium. Here we report new insights into the mechanism underlying this effect, which demonstrates equivalent amplitude in the following models: i) cells expressing a full length PECAM-1 and mutant form of PECAM-1 unable to form homodimers; ii) isolated fractions of cellular membranes; and, iii) immobilized recombinant PECAM-1. These results indicate that CEPAL is mediated not by interference in cellular functions or homophilic PECAM-1 interactions, but rather by conformational changes within the cell adhesion molecule induced by ligand binding. This mechanism, mediated by exposure of partially occult epitopes, is likely to occur in molecules other than PECAM-1 and may represent a generalizable phenomenon with valuable practical applications.

  15. Small Molecule Inhibition of cAMP Response Element Binding Protein in Human Acute Myeloid Leukemia Cells

    PubMed Central

    Mitton, Bryan; Chae, Hee-Don; Hsu, Katie; Dutta, Ritika; Aldana-Masangkay, Grace; Ferrari, Roberto; Davis, Kara; Tiu, Bruce C.; Kaul, Arya; Lacayo, Norman; Dahl, Gary; Xie, Fuchun; Li, Bingbing X.; Breese, Marcus R.; Landaw, Elliot M.; Nolan, Garry; Pellegrini, Matteo; Romanov, Sergei; Xiao, Xiangshu; Sakamoto, Kathleen M.

    2016-01-01

    The transcription factor CREB (cAMP Response Element Binding Protein) is overexpressed in the majority of acute myeloid leukemia (AML) patients, and this is associated with a worse prognosis. Previous work revealed that CREB overexpression augmented AML cell growth, while CREB knockdown disrupted key AML cell functions in vitro. In contrast, CREB knockdown had no effect on long-term hematopoietic stem cell activity in mouse transduction/transplantation assays. Together, these studies position CREB as a promising drug target for AML. To test this concept, a small molecule inhibitor of CREB, XX-650-23, was developed. This molecule blocks a critical interaction between CREB and its required co-activator CBP (CREB Binding Protein), leading to disruption of CREB-driven gene expression. Inhibition of CBP-CREB interaction induced apoptosis and cell cycle arrest in AML cells, and prolonged survival in vivo in mice injected with human AML cells. XX-650-23 had little toxicity on normal human hematopoietic cells and tissues in mice. To understand the mechanism of XX-650-23, we performed RNA-seq, ChIP-seq and Cytometry Time of Flight with human AML cells. Our results demonstrate that small molecule inhibition of CBP-CREB interaction mostly affects apoptotic, cell cycle, and survival pathways, which may represent a novel approach for AML therapy. PMID:27211267

  16. Cloning of a novel phosphotyrosine binding domain containing molecule, Odin, involved in signaling by receptor tyrosine kinases.

    PubMed

    Pandey, Akhilesh; Blagoev, Blagoy; Kratchmarova, Irina; Fernandez, Minerva; Nielsen, Mogens; Kristiansen, Troels Zakarias; Ohara, Osamu; Podtelejnikov, Alexandre V; Roche, Serge; Lodish, Harvey F; Mann, Matthias

    2002-11-14

    We have used a proteomic approach using mass spectrometry to identify signaling molecules involved in receptor tyrosine kinase signaling pathways. Using affinity purification by anti-phosphotyrosine antibodies to enrich for tyrosine phosphorylated proteins, we have identified a novel signaling molecule in the epidermal growth factor receptor signaling pathway. This molecule, designated Odin, contains several ankyrin repeats, two sterile alpha motifs and a phosphotyrosine binding domain and is ubiquitously expressed. Using antibodies against endogenous Odin, we show that it undergoes tyrosine phosphorylation upon addition of growth factors such as EGF or PDGF but not by cytokines such as IL-3 or erythropoietin. Immunofluorescence experiments as well as Western blot analysis on subcellular fractions demonstrated that Odin is localized to the cytoplasm both before and after growth factor treatment. Deletion analysis showed that the phosphotyrosine binding domain of Odin is not required for its tyrosine phosphorylation. Overexpression of Odin, but not an unrelated adapter protein, Grb2, inhibited EGF-induced activation of c-Fos promoter. Microinjection of wild-type or a mutant version lacking the PTB domain into NIH3T3 fibroblasts inhibited PDGF-induced mitogenesis. Taken together, our results indicate that Odin may play a negative role in growth factor receptor signaling pathways.

  17. Assistance of the iron porphyrin ligands to the binding interaction between the Fe center and small molecules in solution.

    PubMed

    Xiao, Jie; Golnak, Ronny; Atak, Kaan; Pflüger, Mika; Pohl, Marvin; Suljoti, Edlira; Winter, Bernd; Aziz, Emad F

    2014-08-07

    Solute-solvent electronic structure interactions of iron porphyrin at very low concentration in dichloromethane (CH2Cl2) liquid solution are reported. Two iron porphyrin complexes are investigated here-iron octaethylporphyrin chloride (FeOEP-Cl) and iron tetraphenylporphyrin chloride (FeTPP-Cl)-using X-ray absorption and emission spectroscopy at the Fe L2,3 edge, and spectra are interpreted with the help of density functional theory/restricted open-shell configuration interaction singles (DFT/ROCIS) calculations. It is argued that the Fe center of FeOEP-Cl is more capable of binding small solvent molecules, exemplified here for Cl2CH2, than FeTPP-Cl in solution. The proposed binding mechanism is through the assistance of the dipole interaction between the porphyrin-ligand system and the solvent molecule, in a situation where the ligand structure and arrangement maximize the binding interactions. Our studies demonstrate that even small ligands, depending on their structure and arrangement, can have considerable effects on porphyrin's metal center chemistry in liquid solution.

  18. pH dependence and exchange of high and low responder peptides binding to a class II MHC molecule.

    PubMed Central

    Reay, P A; Wettstein, D A; Davis, M M

    1992-01-01

    We have compared the binding kinetics of two antigenic peptides to a soluble class II MHC molecule. One of the peptides provokes a strong T cell response and the other a much weaker one. Both show greatly increased (approximately 40-fold) association rates at pH 5 in comparison to neutral pH, consistent with the low pH environment of late endosomes being most conducive to class II MHC--peptide binding. Interestingly, the weak peptide has a much faster off-rate that is significantly increased at pH 5 and it can be entirely replaced in an exchange reaction by the stronger one. This suggests that one characteristic of immunodominant peptides is that of nearly irreversible binding, such that they will be strongly selected for in the course of class II MHC transit and recycling through endosomal compartments. Modelling the parameters of this peptide exchange also suggests that a large fraction of the GPI-chimeric MHC molecules used in this study are 'empty' with respect to endogenous peptides, or else occupied with extremely weak ones, consistent with their inability to load processed peptides intracellularly. PMID:1379172

  19. Small-Molecule Ligands of Methyl-Lysine Binding Proteins: Optimization of Selectivity for L3MBTL3

    PubMed Central

    James, Lindsey I.; Korboukh, Victoria K.; Krichevsky, Liubov; Baughman, Brandi M.; Herold, J. Martin; Norris, Jacqueline L.; Jin, Jian; Kireev, Dmitri B.; Janzen, William P.; Arrowsmith, Cheryl H.; Frye, Stephen V.

    2013-01-01

    Lysine methylation is a key epigenetic mark, the dysregulation of which is linked to many diseases. Small molecule antagonism of methyl-lysine (Kme) binding proteins that recognize such epigenetic marks can improve our understanding of these regulatory mechanisms and potentially validate Kme binding proteins as drug discovery targets. We previously reported the discovery of 1 (UNC1215), the first potent and selective small molecule chemical probe of a methyl-lysine reader protein, L3MBTL3, which antagonizes the mono- and dimethyl-lysine reading function of L3MBTL3. The design, synthesis, and structure activity relationship studies that led to the discovery of 1 are described herein. These efforts established the requirements for potent L3MBTL3 binding and enabled the design of novel antagonists, such as compound 2 (UNC1679), that maintain in vitro and cellular potency with improved selectivity against other MBT-containing proteins. The antagonists described were also found to effectively interact with unlabeled endogenous L3MBTL3 in cells. PMID:24040942

  20. Small-molecule ligands of methyl-lysine binding proteins: optimization of selectivity for L3MBTL3.

    PubMed

    James, Lindsey I; Korboukh, Victoria K; Krichevsky, Liubov; Baughman, Brandi M; Herold, J Martin; Norris, Jacqueline L; Jin, Jian; Kireev, Dmitri B; Janzen, William P; Arrowsmith, Cheryl H; Frye, Stephen V

    2013-09-26

    Lysine methylation is a key epigenetic mark, the dysregulation of which is linked to many diseases. Small-molecule antagonism of methyl-lysine (Kme) binding proteins that recognize such epigenetic marks can improve our understanding of these regulatory mechanisms and potentially validate Kme binding proteins as drug-discovery targets. We previously reported the discovery of 1 (UNC1215), the first potent and selective small-molecule chemical probe of a methyl-lysine reader protein, L3MBTL3, which antagonizes the mono- and dimethyl-lysine reading function of L3MBTL3. The design, synthesis, and structure-activity relationship studies that led to the discovery of 1 are described herein. These efforts established the requirements for potent L3MBTL3 binding and enabled the design of novel antagonists, such as compound 2 (UNC1679), that maintain in vitro and cellular potency with improved selectivity against other MBT-containing proteins. The antagonists described were also found to effectively interact with unlabeled endogenous L3MBTL3 in cells.

  1. 111In-benzyl-DTPA-ZHER2:342, an affibody-based conjugate for in vivo imaging of HER2 expression in malignant tumors.

    PubMed

    Tolmachev, Vladimir; Nilsson, Fredrik Y; Widström, Charles; Andersson, Karl; Rosik, Daniel; Gedda, Lars; Wennborg, Anders; Orlova, Anna

    2006-05-01

    Data on expression of the HER2 (erbB-2) receptor in breast carcinoma make it possible to select the most efficient treatment. There are strong indications that HER2 expression possesses prognostic and predictive values in ovarian, prostate, and lung carcinomas as well. Visualization of HER2 expression using radionuclide targeting can provide important diagnostic information. The Affibody Z(HER2:342) is a short (approximately 7 kDa) phage-display-selected protein that binds HER2 with an affinity of 22 pmol/L. The goal of this study was to evaluate whether (111)In-labeled HER2:342 can be used for imaging of HER2 overexpression in vivo. Z(HER2:342) was labeled with (111)In via isothiocyanate-benzyl-DTPA (DTPA is diethylenetriaminepentaacetic acid) and the conjugate was characterized in vitro and in vivo. (111)In-Benzyl-DTPA-Z(HER2:342) preserved the capacity to bind living HER2-expressing cells specifically. The affinity of In-benzyl-DTPA-Z(HER2:342) to HER2 was 21 pmol/L according to surface plasmon resonance measurements. In nude mice bearing HER2-expressing SKOV-3 xenografts, a tumor uptake of 12% +/- 3% injected activity per gram and a tumor-to-blood ratio of about 100 were obtained 4 h after injection. Tumor uptake in vivo was receptor specific, as it could be blocked with an excess of nonlabeled Z(HER2:342). HER2-expressing xenografts were clearly imaged 4 h after injection using a gamma-camera. (111)In-Benzyl-DTPA-Z(HER2:342) is a promising candidate for visualization of HER2 expression in carcinomas, using the single-photon detection technique.

  2. EQUILIBRIUM AND DYNAMIC DESIGN PRINCIPLES FOR BINDING MOLECULES ENGINEERED FOR REAGENTLESS BIOSENSORS

    PubMed Central

    de Picciotto, Seymour; Imperiali, Barbara; Griffith, Linda G.; Wittrup, K. Dane

    2014-01-01

    Reagentless biosensors rely on the interaction of a binding partner and its target to generate a change in fluorescent signal using an environment sensitive fluorophore or Förster Resonance Energy Transfer. Binding affinity can exert a significant influence on both the equilibrium and the dynamic response characteristics of such a biosensor. We here develop a kinetic model for the dynamic performance of a reagentless biosensor. Using a sinusoidal signal for ligand concentration, our findings suggest that it is optimal to use a binding moiety whose equilibrium dissociation constant matches that of the average predicted input signal, while maximizing both the association rate constant and the dissociation rate constant at the necessary ratio to create the desired equilibrium constant. Although practical limitations constrain the attainment of these objectives, the derivation of these design principles provides guidance for improved reagentless biosensor performance and metrics for quality standards in the development of biosensors. These concepts are broadly relevant to reagentless biosensor modalities. PMID:24814226

  3. Equilibrium and dynamic design principles for binding molecules engineered for reagentless biosensors.

    PubMed

    de Picciotto, Seymour; Imperiali, Barbara; Griffith, Linda G; Wittrup, K Dane

    2014-09-01

    Reagentless biosensors rely on the interaction of a binding partner and its target to generate a change in fluorescent signal using an environment-sensitive fluorophore or Förster resonance energy transfer. Binding affinity can exert a significant influence on both the equilibrium and the dynamic response characteristics of such a biosensor. We here develop a kinetic model for the dynamic performance of a reagentless biosensor. Using a sinusoidal signal for ligand concentration, our findings suggest that it is optimal to use a binding moiety whose equilibrium dissociation constant matches that of the average predicted input signal, while maximizing both the association rate constant and the dissociation rate constant at the necessary ratio to create the desired equilibrium constant. Although practical limitations constrain the attainment of these objectives, the derivation of these design principles provides guidance for improved reagentless biosensor performance and metrics for quality standards in the development of biosensors. These concepts are broadly relevant to reagentless biosensor modalities.

  4. Localization of Bacillus thuringiensis Cry1A toxin-binding molecules in gypsy moth larval gut sections using fluorescence microscopy.

    PubMed

    Valaitis, Algimantas P

    2011-10-01

    The microbial insecticide Bacillus thuringiensis (Bt) produces Cry toxins, proteins that bind to the brush border membranes of gut epithelial cells of insects that ingest it, disrupting the integrity of the membranes, and leading to cell lysis and insect death. In gypsy moth, Lymantria dispar, two toxin-binding molecules for the Cry1A class of Bt toxins have been identified: an aminopeptidase N (APN-1) and a 270kDa anionic glycoconjugate (BTR-270). Studies have shown that APN-1 has a relatively weak affinity and a very narrow specificity to Cry1Ac, the only Cry1A toxin that it binds. In contrast, BTR-270 binds all toxins that are active against L. dispar larvae, and the affinities for these toxins to BTR-270 correlate positively with their respective toxicities. In this study, an immunohistochemical approach was coupled with fluorescence microscopy to localize APN-1 and BTR-270 in paraffin embedded midgut sections of L. dispar larvae. The distribution of cadherin and alkaline phosphatase in the gut tissue was also examined. A strong reaction indicative of polyanionic material was detected with alcian blue staining over the entire epithelial brush border, suggesting the presence of acidic glycoconjugates in the microvillar matrix. The Cry1A toxin-binding sites were confined to the apical surface of the gut epithelial cells with intense labeling of the apical tips of the microvilli. APN-1, BTR-270, and alkaline phosphatase were found to be present exclusively along the brush border microvilli along the entire gut epithelium. In contrast, cadherin, detected only in older gypsy moth larvae, was present both in the apical brush border and in the basement membrane anchoring the midgut epithelial cells. The topographical relationship between the Bt Cry toxin-binding molecules BTR-270 and APN-1 and the Cry1A toxin-binding sites that were confined to the apical brush border of the midgut cells is consistent with findings implicating their involvement in the mechanism of the

  5. The binding of CO molecule with small Wn(n = 2-9) clusters: a DFT investigation

    NASA Astrophysics Data System (ADS)

    Sun, Xiyuan; Du, Jiguang

    2014-08-01

    The hybrid DFT functional has been utilized to investigate CO adsorption on small W n ( n = 2-9) clusters. The reactivity-selectivity descriptor Δf( r) is proved to be an appropriate tool for predicting the local adsorption site. Our results indicate that the binding of CO to tungsten cluster prefers terminal adsorption mode except for W3 trimer. The Wiberg bond index can predict the interaction between W atoms and CO molecule to a good accuracy using a linear fit. The dependency on number of adsorbate and charge state of host clusters is also investigated. The high CO coverage seriously weakens the W-CO interaction. The anion cluster reveals the stronger interaction with CO molecule than cation and neutral ones. Moreover, the interesting coverage-dependence is revealed for anion cluster.

  6. Small molecule functional analogs of peptides that inhibit lambda site-specific recombination and bind Holliday junctions.

    PubMed

    Ranjit, Dev K; Rideout, Marc C; Nefzi, Adel; Ostresh, John M; Pinilla, Clemencia; Segall, Anca M

    2010-08-01

    Our lab has isolated hexameric peptides that are structure-selective ligands of Holliday junctions (HJ), central intermediates of several DNA recombination reactions. One of the most potent of these inhibitors, WRWYCR, has shown antibacterial activity in part due to its inhibition of DNA repair proteins. To increase the therapeutic potential of these inhibitors, we searched for small molecule inhibitors with similar activities. We screened 11 small molecule libraries comprising over nine million individual compounds and identified a potent N-methyl aminocyclic thiourea inhibitor that also traps HJs formed during site-specific recombination reactions in vitro. This inhibitor binds specifically to protein-free HJs and can inhibit HJ resolution by RecG helicase, but only showed modest growth inhibition of bacterial with a hyperpermeable outer membrane; nonetheless, this is an important step in developing a functional analog of the peptide inhibitors. Copyright 2010 Elsevier Ltd. All rights reserved.

  7. Comparative analysis of 10 small molecules binding to carbonic anhydrase II by different investigators using Biacore technology.

    PubMed

    Papalia, Giuseppe A; Leavitt, Stephanie; Bynum, Maggie A; Katsamba, Phinikoula S; Wilton, Rosemarie; Qiu, Huawei; Steukers, Mieke; Wang, Siming; Bindu, Lakshman; Phogat, Sanjay; Giannetti, Anthony M; Ryan, Thomas E; Pudlak, Victoria A; Matusiewicz, Katarzyna; Michelson, Klaus M; Nowakowski, Agnes; Pham-Baginski, Anh; Brooks, Jonathan; Tieman, Bryan C; Bruce, Barry D; Vaughn, Michael; Baksh, Michael; Cho, Yun Hee; Wit, Mieke De; Smets, Alexandra; Vandersmissen, Johan; Michiels, Lieve; Myszka, David G

    2006-12-01

    In this benchmark study, 26 investigators were asked to characterize the kinetics and affinities of 10 sulfonamide inhibitors binding to the enzyme carbonic anhydrase II using Biacore optical biosensors. A majority of the participants collected data that could be fit to a 1:1 interaction model, but a subset of the data sets obtained from some instruments were of poor quality. The experimental errors in the k(a), k(d), and K(D) parameters determined for each of the compounds averaged 34, 24, and 37%, respectively. As expected, the greatest variation in the reported constants was observed for compounds with exceptionally weak affinity and/or fast association rates. The binding constants determined using the biosensor correlated well with solution-based titration calorimetry measurements. The results of this study provide insight into the challenges, as well as the level of experimental variation, that one would expect to observe when using Biacore technology for small molecule analyses.

  8. Two types of sugar-binding protein in the labellum of the fly. Putative taste receptor molecules for sweetness

    PubMed Central

    1993-01-01

    Flies have taste cells specifically sensitive to sweetness. It has been suggested that the cells possess two types of receptor sites covering the receptive field of sweetness. By affinity electrophoresis with the site-specific inhibitory polysaccharides, two types of sugar-binding protein were isolated from the labellar extract of the blowfly. These proteins showed consistent sugar-binding specificities and affinities with the two types of receptor sites for sweetness, respectively. The dissociation constant of the protein-sugar complex varies 100-400 mM and the molecular weight of one type of the protein is 27,000, while that of the other is 31,000 or 32,000. Both proteins were water insoluble and were also detected in the isolated chemosensilla. Thus they are probably located on the taste receptor membrane, and the proteins are likely to act as the taste receptor molecules for sweetness in the fly. PMID:8228908

  9. Neuronal cell adhesion molecule contactin/F11 binds to tenascin via its immunoglobulin-like domains

    PubMed Central

    1992-01-01

    Adhesive interactions between neurons and extracellular matrix (ECM) play a key role in neuronal pattern formation. The prominent role played by the extracellular matrix protein tenascin/cytotactin in the development of the nervous system, tied to its abundance, led us to speculate that brain may contain yet unidentified tenascin receptors. Here we show that the neuronal cell adhesion molecule contactin/F11, a member of the immunoglobulin(Ig)-superfamily, is a cell surface ligand for tenascin in the nervous system. Through affinity chromatography of membrane glycoproteins from chick brain on tenascin-Sepharose, we isolated a major cell surface ligand of 135 kD which we identified as contactin/F11 by NH2-terminal sequencing. The binding specificity between contactin/F11 and tenascin was demonstrated in solid-phase assays. Binding of immunopurified 125I-labeled contactin/F11 to immobilized tenascin is completely inhibited by the addition of soluble tenascin or contactin/F11, but not by fibronectin. When the fractionated isoforms of tenascin were used as substrates, contactin/F11 bound preferentially to the 190-kD isoform. This isoform differs in having no alternatively spliced fibronectin type III domains. Our results imply that the introduction of these additional domains in some way disrupts the contactin/F11 binding site on tenascin. To localize the binding site on contactin/F11, proteolytic fragments were generated and characterized by NH2-terminal sequencing. The smallest contactin/F11 fragment which binds tenascin is 45 kD and also begins with the contactin/F11 NH2-terminal sequence. This implies that contactin/F11 binds to tenascin through a site within the first three Ig-domains. PMID:1382076

  10. Identification of glyceraldehyde-3-phosphate dehydrogenase of epithelial cells as a second molecule that binds to Porphyromonas gingivalis fimbriae.

    PubMed

    Sojar, Hakimuddin T; Genco, Robert J

    2005-07-01

    Binding of Porphyromonas gingivalis to the host cells is an essential step in the pathogenesis of periodontal disease. P. gingivalis binds to and invades epithelial cells, and fimbriae are thought to be involved in this process. In our earlier studies, two major epithelial cell components of 40 and 50 kDa were identified as potential fimbrial receptors. Sequencing of a cyanogen bromide digestion fragment of the 50-kDa component resulted in an internal sequence identical to keratin I molecules, and hence this cytokeratin represents one of the epithelial cell receptors for P. gingivalis fimbriae. In this study, the 40-kDa component of KB cells was isolated and its amino-terminal sequence determined. The N-terminal amino sequence was found to be GKVKVGVNGF and showed perfect homology with human glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Furthermore, purified P. gingivalis fimbriae were found to bind to rabbit muscle GAPDH. Antibodies directed against internal peptide 49-68 and 69-90 of fimbrillin were shown to inhibit the binding of P. gingivalis and of fimbriae to epithelial cells. Antibodies against these peptides also inhibited the binding of fimbriae to GAPDH. Our results confirmed that the amino-terminal domain corresponding to amino residues 49-68 of the fimbrillin protein is the major GAPDH binding domain. These studies point to GAPDH as a major receptor for P. gingivalis major fimbriae and, as such, GAPDH likely plays a role in P. gingivalis adherence and colonization of the oral cavity, as well as triggering host cell processes involved in the pathogenesis of P. gingivalis infections.

  11. Quantitative peptide binding motifs for 19 human and mouse MHC class I molecules derived using positional scanning combinatorial peptide libraries

    PubMed Central

    Sidney, John; Assarsson, Erika; Moore, Carrie; Ngo, Sandy; Pinilla, Clemencia; Sette, Alessandro; Peters, Bjoern

    2008-01-01

    Background It has been previously shown that combinatorial peptide libraries are a useful tool to characterize the binding specificity of class I MHC molecules. Compared to other methodologies, such as pool sequencing or measuring the affinities of individual peptides, utilizing positional scanning combinatorial libraries provides a baseline characterization of MHC molecular specificity that is cost effective, quantitative and unbiased. Results Here, we present a large-scale application of this technology to 19 different human and mouse class I alleles. These include very well characterized alleles (e.g. HLA A*0201), alleles with little previous data available (e.g. HLA A*3201), and alleles with conflicting previous reports on specificity (e.g. HLA A*3001). For all alleles, the positional scanning combinatorial libraries were able to elucidate distinct binding patterns defined with a uniform approach, which we make available here. We introduce a heuristic method to translate this data into classical definitions of main and secondary anchor positions and their preferred residues. Finally, we validate that these matrices can be used to identify candidate MHC binding peptides and T cell epitopes in the vaccinia virus and influenza virus systems, respectively. Conclusion These data confirm, on a large scale, including 15 human and 4 mouse class I alleles, the efficacy of the positional scanning combinatorial library approach for describing MHC class I binding specificity and identifying high affinity binding peptides. These libraries were shown to be useful for identifying specific primary and secondary anchor positions, and thereby simpler motifs, analogous to those described by other approaches. The present study also provides matrices useful for predicting high affinity binders for several alleles for which detailed quantitative descriptions of binding specificity were previously unavailable, including A*3001, A*3201, B*0801, B*1501 and B*1503. PMID:18221540

  12. Discovery of new small molecules targeting the vitronectin-binding site of the urokinase receptor that block cancer cell invasion.

    PubMed

    Rea, Vincenza Elena Anna; Lavecchia, Antonio; Di Giovanni, Carmen; Rossi, Francesca Wanda; Gorrasi, Anna; Pesapane, Ada; de Paulis, Amato; Ragno, Pia; Montuori, Nunzia

    2013-08-01

    Besides focusing urokinase (uPA) proteolytic activity on the cell membrane, the uPA receptor (uPAR) is able to bind vitronectin, via a direct binding site. Furthermore, uPAR interacts with other cell surface receptors, such as integrins, receptor tyrosine kinases, and chemotaxis receptors, triggering cell-signaling pathways that promote tumor progression. The ability of uPAR to coordinate binding and degradation of extracellular matrix (ECM) and cell signaling makes it an attractive therapeutic target in cancer. We used structure-based virtual screening (SB-VS) to search for small molecules targeting the uPAR-binding site for vitronectin. Forty-one compounds were identified and tested on uPAR-negative HEK-293 epithelial cells transfected with uPAR (uPAR-293 cells), using the parental cell line transfected with the empty vector (V-293 cells) as a control. Compounds 6 and 37 selectively inhibited uPAR-293 cell adhesion to vitronectin and the resulting changes in cell morphology and signal transduction, without exerting any effect on V-293 cells. Compounds 6 and 37 inhibited uPAR-293 cell binding to vitronectin with IC50 values of 3.6 and 1.2 μmol/L, respectively. Compounds 6 and 37 targeted S88 and R91, key residues for uPAR binding to vitronectin but also for uPAR interaction with the fMLF family of chemotaxis receptors (fMLF-Rs). As a consequence, compounds 6 and 37 impaired uPAR-293 cell migration toward fetal calf serum (FCS), uPA, and fMLF, likely by inhibiting the interaction between uPAR and FPR1, the high affinity fMLF-R. Both compounds blocked in vitro ECM invasion of several cancer cell types, thus representing new promising leads for pharmaceuticals in cancer.

  13. Single-Molecule Tracking and Its Application in Biomolecular Binding Detection.

    PubMed

    Liu, Cong; Liu, Yen-Liang; Perillo, Evan P; Dunn, Andrew K; Yeh, Hsin-Chih

    2016-01-01

    In the past two decades significant advances have been made in single-molecule detection, which enables the direct observation of single biomolecules at work in real time and under physiological conditions. In particular, the development of single-molecule tracking (SMT) microscopy allows us to monitor the motion paths of individual biomolecules in living systems, unveiling the localization dynamics and transport modalities of the biomolecules that support the development of life. Beyond the capabilities of traditional camera-based tracking techniques, state-of-the-art SMT microscopies developed in recent years can record fluorescence lifetime while tracking a single molecule in the 3D space. This multiparameter detection capability can open the door to a wide range of investigations at the cellular or tissue level, including identification of molecular interaction hotspots and characterization of association/dissociation kinetics between molecules. In this review, we discuss various SMT techniques developed to date, with an emphasis on our recent development of the next generation 3D tracking system that not only achieves ultrahigh spatiotemporal resolution but also provides sufficient working depth suitable for live animal imaging. We also discuss the challenges that current SMT techniques are facing and the potential strategies to tackle those challenges.

  14. Single-Molecule Tracking and Its Application in Biomolecular Binding Detection

    PubMed Central

    Liu, Cong; Liu, Yen-Liang; Perillo, Evan P.; Dunn, Andrew K.; Yeh, Hsin-Chih

    2016-01-01

    In the past two decades significant advances have been made in single-molecule detection, which enables the direct observation of single biomolecules at work in real time and under physiological conditions. In particular, the development of single-molecule tracking (SMT) microscopy allows us to monitor the motion paths of individual biomolecules in living systems, unveiling the localization dynamics and transport modalities of the biomolecules that support the development of life. Beyond the capabilities of traditional camera-based tracking techniques, state-of-the-art SMT microscopies developed in recent years can record fluorescence lifetime while tracking a single molecule in the 3D space. This multiparameter detection capability can open the door to a wide range of investigations at the cellular or tissue level, including identification of molecular interaction hotspots and characterization of association/dissociation kinetics between molecules. In this review, we discuss various SMT techniques developed to date, with an emphasis on our recent development of the next generation 3D tracking system that not only achieves ultrahigh spatiotemporal resolution but also provides sufficient working depth suitable for live animal imaging. We also discuss the challenges that current SMT techniques are facing and the potential strategies to tackle those challenges. PMID:27660404

  15. Ion implantation treatment of beads for covalent binding of molecules: application to bioethanol production using thermophilic beta-glucosidase.

    PubMed

    Nosworthy, Neil J; Kondyurin, Alexey; Bilek, Marcela M M; McKenzie, David R

    2014-01-10

    We have achieved plasma immersion ion implantation (PIII) treatment of beads and powders using a specially designed plasma treatment system. This simple one-step production of functionalized beads provides an attractive alternative to current commercial functional beads, for which proteins must be chemically attached using linkers. Using the enzyme beta-glucosidase as an example we show that PIII treatment of polyethylene beads enables covalent binding with increased activity of the enzyme compared to the untreated beads. Covalent binding was confirmed using detergent washing. The covalently immobilized enzyme has a broader pH range over which it has high activity than the enzyme in solution. The stability of the immobilized molecules was examined using reaction rate as a function of temperature and was shown to be significantly higher on the PIII treated beads compared to untreated beads. We attribute the increased enzyme activity on PIII treated beads to increased protein binding density and better retention of conformation. The results of this work are of significance in the production of ethanol using a flow process. Covalent binding to beads allows more robust attachment for high flow rates, high activity, large surface area and a broad operating pH range. Treatment could be easily adapted for a range of applications such as linking drugs, dyes and proteins to particles of an appropriate size. Copyright © 2013 Elsevier Inc. All rights reserved.

  16. The Small Molecule R-(-)-β-O-Methylsynephrine Binds to Nucleoporin 153 kDa and Inhibits Angiogenesis

    PubMed Central

    Kim, Nam Hee; Pham, Ngoc Bich; Quinn, Ronald J.; Shim, Joong Sup; Cho, Hee; Cho, Sung Min; Park, Sung Wook; Kim, Jeong Hun; Seok, Seung Hyeok; Oh, Jong-Won; Kwon, Ho Jeong

    2015-01-01

    R-(-)-β-O-methylsynephrine (OMe-Syn) is a naturally occurring small molecule that was identified in a previous screen as an inhibitor of angiogenesis. In this study, we conducted two animal model experiments to investigate the in vivo antiangiogenic activity of OMe-Syn. OMe-Syn significantly inhibited angiogenesis in a transgenic zebrafish model as well as in a mouse retinopathy model. To elucidate the underlying mechanisms responsible for the antiangiogenic activity of OMe-Syn, we used phage display cloning to isolate potential OMe-Syn binding proteins from human cDNA libraries and identified nucleoporin 153 kDa (NUP153) as a primary binding partner of OMe-Syn. OMe-Syn competitively inhibited mRNA binding to the RNA-binding domain of NUP153. Furthermore, depletion of NUP153 in human cells or zebrafish embryos led to an inhibition of angiogenesis, in a manner similar to that seen in response to OMe-Syn treatment. These data suggest that OMe-Syn is a promising candidate for the development of a novel antiangiogenic agent and that inhibition of NUP153 is possibly responsible for the antiangiogenic activity of OMe-Syn. PMID:26221075

  17. Screening of the binding of small molecules to proteins by desorption electrospray ionization mass spectrometry combined with protein microarray.

    PubMed

    Yao, Chenxi; Wang, Tao; Zhang, Buqing; He, Dacheng; Na, Na; Ouyang, Jin

    2015-11-01

    The interaction between bioactive small molecule ligands and proteins is one of the important research areas in proteomics. Herein, a simple and rapid method is established to screen small ligands that bind to proteins. We designed an agarose slide to immobilize different proteins. The protein microarrays were allowed to interact with different small ligands, and after washing, the microarrays were screened by desorption electrospray ionization mass spectrometry (DESI MS). This method can be applied to screen specific protein binding ligands and was shown for seven proteins and 34 known ligands for these proteins. In addition, a high-throughput screening was achieved, with the analysis requiring approximately 4 s for one sample spot. We then applied this method to determine the binding between the important protein matrix metalloproteinase-9 (MMP-9) and 88 small compounds. The molecular docking results confirmed the MS results, demonstrating that this method is suitable for the rapid and accurate screening of ligands binding to proteins. Graphical Abstract ᅟ.

  18. The synthesis of multifunctional nanoparticles conjugated with anti-Her2 affibody and monomethylauristatin E

    NASA Astrophysics Data System (ADS)

    Pala, Katarzyna; Jakimowicz, Piotr; Cyranka-Czaja, Anna; Otlewski, Jacek

    2015-04-01

    Conjugation of bioactive xenobiotics with innert particles often improves their efficacy and/or specificity. In this work we designed superparamagnetic ferric oxide nanoparticles (NPs) conjugated with a strong cytotoxic drug, monomethylauristatin E (MMAE), and evaluated their potential against cancer cells. Cytotoxicity tests showed that the conjugate was at least twice as toxic as the free drug. We then studied the cytotoxic potential of the conjugate at an elevated temperature achieved due to the superparamagnetic properties of the NPs, finding no enhancement of cytotoxicity in comparison with that at 37 °C. Next, multifunctional NPs containing MMAE and a targeting agent were synthesized. The targeting agent was the ZHer2:342 affibody specific to Her2 receptor. The selectivity and effectiveness of the conjugates was evaluated using SK-BR3 (Her2-positive) and U-87 MG (a negative control) cell lines. The multifunctional NPs selectively decrease of the viability of the SK-BR3 cells, showing their specificity towards cells overexpressing the Her2 receptor.

  19. Binding motifs of copolymer 1 to multiple sclerosis- and rheumatoid arthritis-associated HLA-DR molecules.

    PubMed

    Fridkis-Hareli, M; Neveu, J M; Robinson, R A; Lane, W S; Gauthier, L; Wucherpfennig, K W; Sela, M; Strominger, J L

    1999-04-15

    Copolymer 1 (Cop 1, poly (Y, E, A, K)) is a random synthetic amino acid copolymer effective in the treatment of relapsing forms of multiple sclerosis (MS). Cop 1 binds promiscuously, with high affinity and in a peptide-specific manner to purified MS-associated HLA-DR2 (DRB1*1501) and rheumatoid arthritis-associated HLA-DR1 (DRB1*0101) or HLA-DR4 (DRB1*0401) molecules. In the present work at least 95% of added Cop 1 could be bound to recombinant "empty" HLA-DR1 and -DR4, and 80% could be bound to HLA-DR2 proteins. Amino acid composition, HPLC profiles, and sequencing patterns of Cop 1 eluted by acid extraction from HLA-DR molecules were similar to those of the unseparated Cop 1. Protruding N-terminal ends of Cop 1 bound to HLA-DR1, -DR2, or -DR4 molecules were then treated with aminopeptidase I, followed by elution, HPLC, and pool sequencing. In contrast to untreated or unbound Cop 1, this material exhibited distinct motifs at some positions with increases in levels of E at the first and second cycles, of K at the second and third cycles, and of Y (presumably at P1 of the bound peptide) at the third to fifth cycles, regardless of the HLA-DR molecule employed. No preference was seen at the following cycles that were mainly A. These first pooled HLA-DR binding epitopes provide clues to the components of Cop 1 that are biologically active in suppressing MS and possibly rheumatoid arthritis.

  20. Cucurbit[8]uril-Containing Multilayer Films for the Photocontrolled Binding and Release of a Guest Molecule.

    PubMed

    Nicolas, Henning; Yuan, Bin; Zhang, Xi; Schönhoff, Monika

    2016-03-15

    The powerful host-guest chemistry of cucurbit[8]uril (CB[8]) was employed to obtain photoresponsive polyelectrolyte multilayer films for the reversible and photocontrolled binding and release of an organic guest molecule. For this purpose, we designed and synthesized a polyelectrolyte with azobenzene side groups. Then, CB[8] was associated with the azo side group to obtain a supramolecular host-guest complex that was further used as building block in order to prepare photoresponsive and CB[8]-containing polyelectrolyte multilayer films. Ultraviolet spectroscopy and a dissipative quartz crystal microbalance are employed to monitor the formation of the host-guest complex and the layer-by-layer self-assembly of the multilayer films, respectively. We demonstrate that the photoresponsive properties of the azo side groups are maintained before and after host-guest complexation with CB[8] in solution and within the multilayer films, respectively. A guest molecule was then specifically included as second binding partner into the CB[8]-containing multilayer films. Subsequently, the release of the guest was performed by UV light irradiation due to the trans-cis isomerization of the adjacent azo side groups. Re-isomerization of the azo side groups was achieved by VIS light irradiation and enabled the rebinding of the guest into CB[8]. Finally, we demonstrate that the photocontrolled binding and release within CB[8]-containing multilayer films can reliably and reversibly be performed over a period of more than 2 weeks with constant binding efficiency. Therefore, we expect such novel type of photosensitive films to have promising future applications in the field of stimuli-responsive nanomaterials.

  1. Binding of atoms and stability of molecules in Hartree and Thomas-Fermi type theories

    SciTech Connect

    Catto, I. ); Lions, P.L.

    1993-01-01

    This paper is the sequel of a previous work where the authors showed a general necessary and sufficient condition for the stability of an arbitrary molecular system (possibly ionized) in the framework of Hartree or Thomas-Fermi type theories. This condition, roughly speaking, meant that certain particular subsystems have to be bound. They show here in particular that this condition reduces for general molecular system with nonnegative excess charge to the binding of all subsystems with the same property. For neutral molecular systems, this reduces to the binding of all neutral subsystems. In both cases, all other subsystems can be bound. They also show that, for the Hartree-Fock and Hartree models, this condition involves only physical subsystems. They use these reduced conditions to conclude about the stability or the binding in some particular cases. This work is also the second of a series devoted to these equations and the authors come back on the binding of neutral systems in Part 3. 18 refs.

  2. Peptide-binding motifs of two common equine class I MHC molecules in Thoroughbred horses.

    PubMed

    Bergmann, Tobias; Lindvall, Mikaela; Moore, Erin; Moore, Eugene; Sidney, John; Miller, Donald; Tallmadge, Rebecca L; Myers, Paisley T; Malaker, Stacy A; Shabanowitz, Jeffrey; Osterrieder, Nikolaus; Peters, Bjoern; Hunt, Donald F; Antczak, Douglas F; Sette, Alessandro

    2017-05-01

    Quantitative peptide-binding motifs of MHC class I alleles provide a valuable tool to efficiently identify putative T cell epitopes. Detailed information on equine MHC class I alleles is still very limited, and to date, only a single equine MHC class I allele, Eqca-1*00101 (ELA-A3 haplotype), has been characterized. The present study extends the number of characterized ELA class I specificities in two additional haplotypes found commonly in the Thoroughbred breed. Accordingly, we here report quantitative binding motifs for the ELA-A2 allele Eqca-16*00101 and the ELA-A9 allele Eqca-1*00201. Utilizing analyses of endogenously bound and eluted ligands and the screening of positional scanning combinatorial libraries, detailed and quantitative peptide-binding motifs were derived for both alleles. Eqca-16*00101 preferentially binds peptides with aliphatic/hydrophobic residues in position 2 and at the C-terminus, and Eqca-1*00201 has a preference for peptides with arginine in position 2 and hydrophobic/aliphatic residues at the C-terminus. Interestingly, the Eqca-16*00101 motif resembles that of the human HLA A02-supertype, while the Eqca-1*00201 motif resembles that of the HLA B27-supertype and two macaque class I alleles. It is expected that the identified motifs will facilitate the selection of candidate epitopes for the study of immune responses in horses.

  3. A Small Molecule Inhibitor of Pot1 Binding to Telomeric DNA

    PubMed Central

    Altschuler, Sarah E.; Croy, Johnny E.; Wuttke, Deborah S.

    2012-01-01

    Chromosome ends are complex structures, consisting of repetitive DNA sequence terminating in an ssDNA overhang with many associated proteins. Because alteration of these ends is a hallmark of cancer, telomeres and telomere maintenance have been prime drug targets. The universally conserved ssDNA overhang is sequence-specifically bound and regulated by Pot1 (protection of telomeres), and perturbation of Pot1 function has deleterious effects for proliferating cells. The specificity of the Pot1/ssDNA interaction and the key involvement of this protein in telomere maintenance have suggested directed inhibition of Pot1/ssDNA binding as an efficient means of disrupting telomere function. To explore this idea, we developed a high-throughput time-resolved fluorescence resonance energy transfer (TR-FRET) screen for inhibitors of Pot1/ssDNA interaction. We conducted this screen with the DNA-binding subdomain of S. pombe Pot1 (Pot1pN), which confers the vast majority of Pot1 sequence-specificity and is highly similar to the first domain of human Pot1 (hPOT1). Screening a library of ~20,000 compounds yielded a single inhibitor, which we found interacted tightly with submicromolar affinity. Furthermore, this compound, subsequently identified as the bis-azo dye Congo red, was able to competitively inhibit hPOT1 binding to telomeric DNA. ITC and NMR chemical shift analysis suggest that CR interacts specifically with the ssDNA-binding cleft of Pot1, and that alteration of this surface disrupts CR binding. The identification of a specific inhibitor of ssDNA interaction establishes a new pathway for targeted telomere disruption. PMID:22978652

  4. L1 adhesion molecule on mouse leukocytes: regulation and involvement in endothelial cell binding.

    PubMed

    Hubbe, M; Kowitz, A; Schirrmacher, V; Schachner, M; Altevogt, P

    1993-11-01

    L1 is a cell surface glycoprotein of the immunoglobulin superfamily which was initially shown to mediate adhesion between neural cells. Recently we have reported that L1 is expressed by bone marrow cells and the majority of mature lymphocytes (Kowitz et al., Eur. J. Immunol. 1992. 22: 1199-1205). To analyze the function of L1 on leukocytes we studied its regulation following cell activation. In vitro activation of B lymphocytes with lipopolysaccharide or T lymphocytes with phorbol 12-myristate 13-acetate/Ca2+ ionophore, concanavalin A or anti-CD3 monoclonal antibody as well as in vivo activation of V beta 8+ T cells with staphylococcal enterotoxin B (SEB) revealed a down-regulation of L1 within 48 h. A rapid loss of L1 expression was seen when mouse neutrophils were activated with PMA alone. This rapid loss paralleled the shedding of L-selectin. We also studied a possible role of L1 in the binding of leukocytes to endothelial cells. ESb-MP lymphoma cells with a high expression of L1 (L1hi) could bind to bend3 endothelioma cells without prior activation with inflammatory cytokines. The interaction was inhibited by anti-L1 antibodies. In contrast, ESb-MP cells with low L1 expression (L1lo) were only marginally bound. Latex beads coated with affinity-isolated L1 antigen were also able to bind to the endothelioma cells in a specific fashion. The binding of ESb-MP lymphoma cells required Ca2+ and Mg2+ ions and was sensitive to cold temperature. Since the endothelioma cells did not express L1 the binding mechanism studied here is distinct from the established L1-L1 homotypic interaction. It is possible that the novel L1-mediated adhesion pathway involves an unidentified ligand and could play a role in leukocyte migration.

  5. Regulation of DNA Metabolism by DNA-Binding Proteins Probed by Single Molecule Spectroscopy

    DTIC Science & Technology

    2006-12-05

    denaturation The Watson - Crick double- helix is the thermodynamically stable configuration of a DNA molecule under physiological conditions (normal salt and...room/body temperature). This stability is effected (a) by Watson - Crick H-bonding, that is essential for the specificity of base pairing, i.e., for...guarantees the high level of fidelity during replication and transcription. (b) The second contribution to DNA helix stability comes from base-stacking

  6. Extended Lagrangian Density Functional Tight-Binding Molecular Dynamics for Molecules and Solids

    SciTech Connect

    Aradi, Bálint; Niklasson, Anders M. N.; Frauenheim, Thomas

    2015-06-26

    A computationally fast quantum mechanical molecular dynamics scheme using an extended Lagrangian density functional tight-binding formulation has been developed and implemented in the DFTB+ electronic structure program package for simulations of solids and molecular systems. The scheme combines the computational speed of self-consistent density functional tight-binding theory with the efficiency and long-term accuracy of extended Lagrangian Born–Oppenheimer molecular dynamics. Furthermore, for systems without self-consistent charge instabilities, only a single diagonalization or construction of the single-particle density matrix is required in each time step. The molecular dynamics simulation scheme can also be applied to a broad range of problems in materials science, chemistry, and biology.

  7. Identification of small molecules that bind to the mitochondrial protein mitoNEET.

    PubMed

    Geldenhuys, Werner J; Yonutas, Heather M; Morris, Daniel L; Sullivan, Patrick G; Darvesh, Altaf S; Leeper, Thomas C

    2016-11-01

    MitoNEET (CISD1) is a 2Fe-2S iron-sulfur cluster protein belonging to the zinc-finger protein family. Recently mitoNEET has been shown to be a major role player in the mitochondrial function associated with metabolic type diseases such as obesity and cancers. The anti-diabetic drug pioglitazone and rosiglitazone were the first identified ligands to mitoNEET. Since little is known about structural requirements for ligand binding to mitoNEET, we screened a small set of compounds to gain insight into these requirements. We found that the thiazolidinedione (TZD) warhead as seen in rosiglitazone was not an absolutely necessity for binding to mitoNEET. These results will aid in the development of novel compounds that can be used to treat mitochondrial dysfunction seen in several diseases.

  8. Extended Lagrangian Density Functional Tight-Binding Molecular Dynamics for Molecules and Solids.

    PubMed

    Aradi, Bálint; Niklasson, Anders M N; Frauenheim, Thomas

    2015-07-14

    A computationally fast quantum mechanical molecular dynamics scheme using an extended Lagrangian density functional tight-binding formulation has been developed and implemented in the DFTB+ electronic structure program package for simulations of solids and molecular systems. The scheme combines the computational speed of self-consistent density functional tight-binding theory with the efficiency and long-term accuracy of extended Lagrangian Born-Oppenheimer molecular dynamics. For systems without self-consistent charge instabilities, only a single diagonalization or construction of the single-particle density matrix is required in each time step. The molecular dynamics simulation scheme can be applied to a broad range of problems in materials science, chemistry, and biology.

  9. Extended Lagrangian Density Functional Tight-Binding Molecular Dynamics for Molecules and Solids

    DOE PAGES

    Aradi, Bálint; Niklasson, Anders M. N.; Frauenheim, Thomas

    2015-06-26

    A computationally fast quantum mechanical molecular dynamics scheme using an extended Lagrangian density functional tight-binding formulation has been developed and implemented in the DFTB+ electronic structure program package for simulations of solids and molecular systems. The scheme combines the computational speed of self-consistent density functional tight-binding theory with the efficiency and long-term accuracy of extended Lagrangian Born–Oppenheimer molecular dynamics. Furthermore, for systems without self-consistent charge instabilities, only a single diagonalization or construction of the single-particle density matrix is required in each time step. The molecular dynamics simulation scheme can also be applied to a broad range of problems in materialsmore » science, chemistry, and biology.« less

  10. Efficient Computation of Small-Molecule Configurational Binding Entropy and Free Energy Changes by Ensemble Enumeration

    PubMed Central

    2013-01-01

    Here we present a novel, end-point method using the dead-end-elimination and A* algorithms to efficiently and accurately calculate the change in free energy, enthalpy, and configurational entropy of binding for ligand–receptor association reactions. We apply the new approach to the binding of a series of human immunodeficiency virus (HIV-1) protease inhibitors to examine the effect ensemble reranking has on relative accuracy as well as to evaluate the role of the absolute and relative ligand configurational entropy losses upon binding in affinity differences for structurally related inhibitors. Our results suggest that most thermodynamic parameters can be estimated using only a small fraction of the full configurational space, and we see significant improvement in relative accuracy when using an ensemble versus single-conformer approach to ligand ranking. We also find that using approximate metrics based on the single-conformation enthalpy differences between the global minimum energy configuration in the bound as well as unbound states also correlates well with experiment. Using a novel, additive entropy expansion based on conditional mutual information, we also analyze the source of ligand configurational entropy loss upon binding in terms of both uncoupled per degree of freedom losses as well as changes in coupling between inhibitor degrees of freedom. We estimate entropic free energy losses of approximately +24 kcal/mol, 12 kcal/mol of which stems from loss of translational and rotational entropy. Coupling effects contribute only a small fraction to the overall entropy change (1–2 kcal/mol) but suggest differences in how inhibitor dihedral angles couple to each other in the bound versus unbound states. The importance of accounting for flexibility in drug optimization and design is also discussed. PMID:24250277

  11. Accessibility of adenine binding sites in dehydrogenases to small molecules studied by fluorescence quenching.

    PubMed

    Gafni, A

    1979-04-17

    Quenching of the fluorescence of ethenoadenine derivatives by iodide ions and by methionine was studied in solution and when the nucleotides were bound to several dehydrogenases. The fluorescence of epsilonADPR in neutral aqueous solution is dynamically quenched by both quenching agents. The quenching of free epsilonNAD+ by methionine was found to be predominantly static and was satisfactorily described to result from complex formation between quencher and dinucleotide. The rat constant for quenching by iodide of epsilonNAD+ in the ternary complex with LADH and pyrazole is comparable to that of free epsilonADPR or epsilonADP. it is concluded that the bound epsilon-adenine ring is partially exposed to the solvent. The opening, to the solvent, of the adenine binding site is not large enough to allow free methionine diffusion since the rate constant for quenching of bound coenzyme by this quenching agent is relatively small. The difference between the rate constants for quenching of free and enzyme bound nucleotide was used to evaluate the binding constants of epsilonADPR to GPDH, epsilonNAD+ to LDH, and oxalate to the LDH:epsilonNAD+ complex. This technique may prove to be particularly useful when the binding of a fluorescent ligand to a protein is not accompanied by significant changes in its fluorescence.

  12. Discovery of a small-molecule HIV-1 integrase inhibitor-binding site

    PubMed Central

    Al-Mawsawi, Laith Q.; Fikkert, Valery; Dayam, Raveendra; Witvrouw, Myriam; Burke, Terrence R.; Borchers, Christoph H.; Neamati, Nouri

    2006-01-01

    Herein, we report the identification of a unique HIV-1 integrase (IN) inhibitor-binding site using photoaffinity labeling and mass spectrometric analysis. We chemically incorporated a photo-activatable benzophenone moiety into a series of coumarin-containing IN inhibitors. A representative of this series was covalently photo-crosslinked with the IN core domain and subjected to HPLC purification. Fractions were subsequently analyzed by using MALDI-MS and electrospray ionization (ESI)-MS to identify photo-crosslinked products. In this fashion, a single binding site for an inhibitor located within the tryptic peptide 128AACWWAGIK136 was identified. Site-directed mutagenesis followed by in vitro inhibition assays resulted in the identification of two specific amino acid residues, C130 and W132, in which substitutions resulted in a marked resistance to the IN inhibitors. Docking studies suggested a specific disruption in functional oligomeric IN complex formation. The combined approach of photo-affinity labeling/MS analysis with site-directed mutagenesis/molecular modeling is a powerful approach for elucidating inhibitor-binding sites of proteins at the atomic level. This approach is especially important for the study of proteins that are not amenable to traditional x-ray crystallography and NMR techniques. This type of structural information can help illuminate processes of inhibitor resistance and thereby facilitate the design of more potent second-generation inhibitors. PMID:16785440

  13. Pocketome: an encyclopedia of small-molecule binding sites in 4D.

    PubMed

    Kufareva, Irina; Ilatovskiy, Andrey V; Abagyan, Ruben

    2012-01-01

    The importance of binding site plasticity in protein-ligand interactions is well-recognized, and so are the difficulties in predicting the nature and the degree of this plasticity by computational means. To assist in understanding the flexible protein-ligand interactions, we constructed the Pocketome, an encyclopedia of about one thousand experimentally solved conformational ensembles of druggable binding sites in proteins, grouped by location and consistent chain/cofactor composition. The multiplicity of pockets within the ensembles adds an extra, fourth dimension to the Pocketome entry data. Within each ensemble, the pockets were carefully classified by the degree of their pairwise similarity and compatibility with different ligands. The core of the Pocketome is derived regularly and automatically from the current releases of the Protein Data Bank and the Uniprot Knowledgebase; this core is complemented by entries built from manually provided seed ligand locations. The Pocketome website (www.pocketome.org) allows searching for the sites of interest, analysis of conformational clusters, important residues, binding compatibility matrices and interactive visualization of the ensembles using the ActiveICM web browser plugin. The Pocketome collection can be used to build multi-conformational docking and 3D activity models as well as to design cross-docking and virtual ligand screening benchmarks.

  14. A single-step competitive binding assay for mapping of single DNA molecules.

    PubMed

    Nyberg, Lena K; Persson, Fredrik; Berg, Johan; Bergström, Johanna; Fransson, Emelie; Olsson, Linnea; Persson, Moa; Stålnacke, Antti; Wigenius, Jens; Tegenfeldt, Jonas O; Westerlund, Fredrik

    2012-01-06

    Optical mapping of genomic DNA is of relevance for a plethora of applications such as scaffolding for sequencing and detection of structural variations as well as identification of pathogens like bacteria and viruses. For future clinical applications it is desirable to have a fast and robust mapping method based on as few steps as possible. We here demonstrate a single-step method to obtain a DNA barcode that is directly visualized using nanofluidic devices and fluorescence microscopy. Using a mixture of YOYO-1, a bright DNA dye, and netropsin, a natural antibiotic with very high AT specificity, we obtain a DNA map with a fluorescence intensity profile along the DNA that reflects the underlying sequence. The netropsin binds to AT-tetrads and blocks these binding sites from YOYO-1 binding which results in lower fluorescence intensity from AT-rich regions of the DNA. We thus obtain a DNA barcode that is dark in AT-rich regions and bright in GC-rich regions with kilobasepair resolution. We demonstrate the versatility of the method by obtaining a barcode on DNA from the phage T4 that captures its circular permutation and agrees well with its known sequence. Copyright © 2011 Elsevier Inc. All rights reserved.

  15. Binding site identification and role of permanent water molecule of PIM-3 kinase: A molecular dynamics study.

    PubMed

    Ul-Haq, Zaheer; Gul, Sana; Usmani, Saman; Wadood, Abdul; Khan, Waqasuddin

    2015-11-01

    The kinome is a protein kinase complement of the human genome, categorized as serine/threonine and tyrosine kinases. These kinases catalyze phosphorylation reaction by using ATP as phosphoryl donor. Proviral Integration Site for Moloney Murine Leukemia Virus (PIM) kinase encodes serine/threonine protein kinases that recognized as proto-oncogene, responsible for rapid growth of cancerous cells. It is implicated in cell survival and function via cell cycle progression and its metabolism. PIM-3, sub-member of PIM kinases is a proto-oncogene, its overexpression inhibits apoptosis, and results in progression of hepatocellular carcinoma. PIM-3 is considered as a promising drug target but attempts to develop its specific inhibitors is slowed down due to the lack of 3D structure by any experimental technique. In silico techniques generally facilitate scientist to explore hidden structural features in order to improve drug discovery. In the present study, homology modeling, molecular docking and MD simulation techniques were utilized to explore the structure and dynamics of PIM-3 kinase. Induction of water molecules during molecular docking simulation explored differences in the hinge region between PIM-1 and PIM-3 kinases that may be responsible for specificity. Furthermore, role of water molecules in the active site was also explored via radial distribution function (RDF) after a 10 ns molecular dynamics (MD) simulations. Generated RDF plots exhibited the importance of water for inhibitor binding through their bridging capability that links the ligand with binding site residues.

  16. Binding and movement of individual Cel7A cellobiohydrolases on crystalline cellulose surfaces revealed by single-molecule fluorescence imaging.

    PubMed

    Jung, Jaemyeong; Sethi, Anurag; Gaiotto, Tiziano; Han, Jason J; Jeoh, Tina; Gnanakaran, Sandrasegaram; Goodwin, Peter M

    2013-08-16

    The efficient catalytic conversion of biomass to bioenergy would meet a large portion of energy requirements in the near future. A crucial step in this process is the enzyme-catalyzed hydrolysis of cellulose to glucose that is then converted into fuel such as ethanol by fermentation. Here we use single-molecule fluorescence imaging to directly monitor the movement of individual Cel7A cellobiohydrolases from Trichoderma reesei (TrCel7A) on the surface of insoluble cellulose fibrils to elucidate molecular level details of cellulase activity. The motion of multiple, individual TrCel7A cellobiohydrolases was simultaneously recorded with ∼15-nm spatial resolution. Time-resolved localization microscopy provides insights on the activity of TrCel7A on cellulose and informs on nonproductive binding and diffusion. We measured single-molecule residency time distributions of TrCel7A bound to cellulose both in the presence of and absence of cellobiose the major product and a potent inhibitor of Cel7A activity. Combining these results with a kinetic model of TrCel7A binding provides microscopic insight into interactions between TrCel7A and the cellulose substrate.

  17. Synthesis of Zn-MOF incorporating titanium-hydrides as active sites binding H{sub 2} molecules

    SciTech Connect

    Kim, Jongsik; Ok Kim, Dong; Wook Kim, Dong; Sagong, Kil

    2015-10-15

    This paper describes the synthetic effort for a Zn-MOF imparting Ti-H as a preferential binding site potentially capturing H{sub 2} molecules via Kubas-type interaction. The formation mechanism of Ti-H innate to the final material was potentially demonstrated to follow a radical dissociation rather than a β-hydrogen elimination and a C-H reductive elimination. - Graphical abstract: This study details the synthesis and the formation mechanism of Zn-MOF adsorbent site-isolating TiH{sub 3} that can potentially capture H{sub 2} molecules via Kubas-binding mechanism. - Highlights: • OH-functionalized Zn-MOF was employed as a reactive template to site-isolate TiH{sub 3}. • This MOF was post-synthetically modified using a tetracyclohexyl titanium (IV). • This intermediate was hydrogenolyzed to change ligand from cyclohexyl to hydride. • Formation mechanism of TiH{sub 3} was investigated via two control GC–MS experiments. • Final Zn-MOF potentially site-isolating TiH{sub 3} species was used as a H{sub 2} adsorbent.

  18. Binding and Movement of Individual Cel7A Cellobiohydrolases on Crystalline Cellulose Surfaces Revealed by Single-molecule Fluorescence Imaging*

    PubMed Central

    Jung, Jaemyeong; Sethi, Anurag; Gaiotto, Tiziano; Han, Jason J.; Jeoh, Tina; Gnanakaran, Sandrasegaram; Goodwin, Peter M.

    2013-01-01

    The efficient catalytic conversion of biomass to bioenergy would meet a large portion of energy requirements in the near future. A crucial step in this process is the enzyme-catalyzed hydrolysis of cellulose to glucose that is then converted into fuel such as ethanol by fermentation. Here we use single-molecule fluorescence imaging to directly monitor the movement of individual Cel7A cellobiohydrolases from Trichoderma reesei (TrCel7A) on the surface of insoluble cellulose fibrils to elucidate molecular level details of cellulase activity. The motion of multiple, individual TrCel7A cellobiohydrolases was simultaneously recorded with ∼15-nm spatial resolution. Time-resolved localization microscopy provides insights on the activity of TrCel7A on cellulose and informs on nonproductive binding and diffusion. We measured single-molecule residency time distributions of TrCel7A bound to cellulose both in the presence of and absence of cellobiose the major product and a potent inhibitor of Cel7A activity. Combining these results with a kinetic model of TrCel7A binding provides microscopic insight into interactions between TrCel7A and the cellulose substrate. PMID:23818525

  19. Bundle formation of smooth muscle desmin intermediate filaments by calponin and its binding site on the desmin molecule.

    PubMed

    Fujii, T; Takagi, H; Arimoto, M; Ootani, H; Ueeda, T

    2000-03-01

    Smooth muscle basic calponin, a major actin-, tropomyosin-, and calmodulin-binding protein, has been examined for its ability to interact with desmin intermediate filaments from smooth muscle cells using sedimentation analysis, turbidity changes, chemical cross-linking, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI TOF/MS), and electron microscopic observations. Calponin interacted with desmin intermediate filaments in a concentration-dependent manner in vitro. The binding of calponin to desmin produced dense aggregates at 30 degrees C. The dense aggregates were observed by electron microscopy to be composed of large anisotropic bundles of desmin filaments, indicating that calponin forms bundles of desmin filaments. The addition of calmodulin or S100 to the mixture of calponin and desmin caused the removal of calponin from the desmin filaments and inhibited bundle formation in the presence of Ca(2+), but not in the presence of EGTA. Calponin-related proteins including G-actin, tropomyosin, and SM22, had little effect on the binding of calponin to desmin filaments, whereas tubulin weakly inhibited the binding. Desmin had little influence on the calponin-actin and calponin-tubulin interactions using the zero-length cross-linker, EDC. Domain mapping with chymotryptic digestion showed that the binding site of calponin resides within the central a-helical rod domain of the desmin molecule. The chemical cross-linked products of calponin and synthetic peptides (TQ27, TNEKVELQELNDRFANYIEKVRFLEQQ; EE24, EEELRELRRQVDALTGQRARVEVE) derived from the rod domain were detected by MALDI TOF/MS. Furthermore, the calponin-desmin interaction was significantly inhibited by the addition of EE24, but only slightly by TQ27. These results suggest that calponin may act as a cross-linking protein between desmin filaments as well as among intermediate filaments, microfilaments and microtubules in smooth muscle cells.

  20. Single-molecule FRET studies of the cooperative and non-cooperative binding kinetics of the bacteriophage T4 single-stranded DNA binding protein (gp32) to ssDNA lattices at replication fork junctions

    PubMed Central

    Lee, Wonbae; Gillies, John P.; Jose, Davis; Israels, Brett A.; von Hippel, Peter H.; Marcus, Andrew H.

    2016-01-01

    Gene 32 protein (gp32) is the single-stranded (ss) DNA binding protein of the bacteriophage T4. It binds transiently and cooperatively to ssDNA sequences exposed during the DNA replication process and regulates the interactions of the other sub-assemblies of the replication complex during the replication cycle. We here use single-molecule FRET techniques to build on previous thermodynamic studies of gp32 binding to initiate studies of the dynamics of the isolated and cooperative binding of gp32 molecules within the replication complex. DNA primer/template (p/t) constructs are used as models to determine the effects of ssDNA lattice length, gp32 concentration, salt concentration, binding cooperativity and binding polarity at p/t junctions. Hidden Markov models (HMMs) and transition density plots (TDPs) are used to characterize the dynamics of the multi-step assembly pathway of gp32 at p/t junctions of differing polarity, and show that isolated gp32 molecules bind to their ssDNA targets weakly and dissociate quickly, while cooperatively bound dimeric or trimeric clusters of gp32 bind much more tightly, can ‘slide’ on ssDNA sequences, and exhibit binding dynamics that depend on p/t junction polarities. The potential relationships of these binding dynamics to interactions with other components of the T4 DNA replication complex are discussed. PMID:27694621

  1. Major versus minor groove DNA binding of a bisarginylporphyrin hybrid molecule: A molecular mechanics investigation

    NASA Astrophysics Data System (ADS)

    Gresh, Nohad; Perrée-fauvet, Martine

    1999-03-01

    On the basis of theoretical computations, we have recently synthesised [Perrée-Fauvet, M. and Gresh, N., Tetrahedron Lett., 36 (1995) 4227] a bisarginyl conjugate of a tricationic porphyrin (BAP), designed to target, in the major groove of DNA, the d(GGC GCC)2 sequence which is part of the primary binding site of the HIV-1 retrovirus site [Wain-Hobson, S. et al., Cell, 40 (1985) 9]. In the theoretical model, the chromophore intercalates at the central d(CpG)2 step and each of the arginyl arms targets O6/N7belonging to guanine bases flanking the intercalation site. Recent IR and UV-visible spectroscopic studies have confirmed the essential features of these theoretical predictions [Mohammadi, S. et al., Biochemistry, 37 (1998) 6165]. In the present study, we compare the energies of competing intercalation modes of BAP to several double-stranded oligonucleotides, according to whether one, two or three N- methylpyridinium rings project into the major groove. Correspondingly, three minor groove binding modes were considered, the arginyl arms now targeting N3, O2 sites belonging to the purine or pyrimidine bases flanking the intercalation site. This investigation has shown that: (i) in both the major and minor grooves, the best-bound complexes have the three N-methylpyridinium rings in the groove opposite to that of the phenyl group bearing the arginyl arms; (ii) major groove binding is preferred over minor groove binding by a significant energy (29 kcal/mol); and (iii) the best-bound sequence in the major groove is d(GGC GCC)2 with two successive guanines upstream from the intercalation. On the other hand, due to the flexibility of the arginyl arms, other GC-rich sequences have close binding energies, two of them being less stable than it by less than 8 kcal/mol. These results serve as the basis for the design of derivatives of BAP with enhanced sequence selectivities in the major groove.

  2. Role of ICAM-1 polymorphisms (G241R, K469E) in mediating its single-molecule binding ability: Atomic force microscopy measurements on living cells

    SciTech Connect

    Bai, Rui; Yi, Shaoqiong; Zhang, Xuejie; Liu, Huiliang; Fang, Xiaohong

    2014-06-13

    Highlights: • We evaluated both single molecule binding ability and expression level of 4 ICAM-1 mutations. • AFM was used to measure single-molecule binding ability on living cells. • The SNP of ICAM-1 may induce changes in expressions rather than single-molecule binding ability. - Abstract: Atherosclerosis (As) is characterized by chronic inflammation and is a major cause of human mortality. ICAM-1-mediated adhesion of leukocytes in vessel walls plays an important role in the pathogenesis of atherosclerosis. Two single nucleotide polymorphisms (SNPs) of human intercellular adhesion molecule-1 (ICAM-1), G241R and K469E, are associated with a number of inflammatory diseases. SNP induced changes in ICAM-1 function rely not only on the expression level but also on the single-molecule binding ability which may be affected by single molecule conformation variations such as protein splicing and folding. Previous studies have shown associations between G241R/K469E polymorphisms and ICAM-1 gene expression. Nevertheless, few studies have been done that focus on the single-molecule forces of the above SNPs and their ligands. In the current study, we evaluated both single molecule binding ability and expression level of 4 ICAM-1 mutations – GK (G241/K469), GE (G241/E469), RK (R241/K469) and RE (R241/E469). No difference in adhesion ability was observed via cell adhesion assay or atomic force microscopy (AFM) measurement when comparing the GK, GE, RK, or RE genotypes of ICAM-1 to each other. On the other hand, flow cytometry suggested that there was significantly higher expression of GE genotype of ICAM-1 on transfected CHO cells. Thus, we concluded that genetic susceptibility to diseases related to ICAM-1 polymorphisms, G241R or K469E, might be due to the different expressions of ICAM-1 variants rather than to the single-molecule binding ability of ICAM-1.

  3. Dileucine and PDZ-binding Motifs Mediate Synaptic Adhesion-like Molecule 1 (SALM1) Trafficking in Hippocampal Neurons*

    PubMed Central

    Seabold, Gail K.; Wang, Philip Y.; Petralia, Ronald S.; Chang, Kai; Zhou, Arthur; McDermott, Mark I.; Wang, Ya-Xian; Milgram, Sharon L.; Wenthold, Robert J.

    2012-01-01

    Synaptic adhesion-like molecules (SALMs) are a family of cell adhesion molecules involved in neurite outgrowth and synapse formation. Of the five family members, only SALM1, -2, and -3 contain a cytoplasmic C-terminal PDZ-binding motif. We have found that SALM1 is unique among the SALMs because deletion of its PDZ-binding motif (SALM1ΔPDZ) blocks its surface expression in heterologous cells. When expressed in hippocampal neurons, SALM1ΔPDZ had decreased surface expression in dendrites and the cell soma but not in axons, suggesting that the PDZ-binding domain may influence cellular trafficking of SALMs to specific neuronal locations. Endoglycosidase H digestion assays indicated that SALM1ΔPDZ is retained in the endoplasmic reticulum (ER) in heterologous cells. However, when the entire C-terminal tail of SALM1 was deleted, SALM1 was detected on the cell surface. Using serial deletions, we identified a region of SALM1 that contains a putative dileucine ER retention motif, which is not present in the other SALMs. Mutation of this DXXXLL motif allowed SALM1 to leave the ER and enhanced its surface expression in heterologous cells and neurons. An increase in the number of protrusions at the dendrites and cell body was observed when this SALM1 mutant was expressed in hippocampal neurons. With electron microscopy, these protrusions appeared to be irregular, enlarged spines and filopodia. Thus, enrichment of SALM1 on the cell surface affects dendritic arborization, and intracellular motifs regulate its dendritic versus axonal localization. PMID:22174418

  4. Critical binding and electron scattering by symmetric-top polar molecules

    SciTech Connect

    Garrett, W. R.

    2014-10-28

    Quantum treatments of electron interactions with polar symmetric-top rotor molecules show features not present in the treatment of the linear-polar-rotor model. For symmetric tops possessing non-zero angular momentum about the symmetry axis, a new critical dipole can be defined that guarantees an infinite set of dipole-bound states independent of the values of the components of the inertial tensor. Additionally, for this same class, the scattering cross section diverges for all nonzero values of dipole moments and inertial moments, similar to solutions for the fixed linear dipole. Additional predictions are presented for electron affinities and rotational resonances of these systems.

  5. Critical binding and electron scattering by symmetric-top polar molecules.

    PubMed

    Garrett, W R

    2014-10-28

    Quantum treatments of electron interactions with polar symmetric-top rotor molecules show features not present in the treatment of the linear-polar-rotor model. For symmetric tops possessing non-zero angular momentum about the symmetry axis, a new critical dipole can be defined that guarantees an infinite set of dipole-bound states independent of the values of the components of the inertial tensor. Additionally, for this same class, the scattering cross section diverges for all nonzero values of dipole moments and inertial moments, similar to solutions for the fixed linear dipole. Additional predictions are presented for electron affinities and rotational resonances of these systems.

  6. Separation of rare gases and chiral molecules by selective binding in porous organic cages

    SciTech Connect

    Chen, Linjiang; Reiss, Paul S.; Chong, Samantha Y.; Holden, Daniel; Jelfs, Kim E.; Hasell, Tom; Little, Marc A.; Kewley, Adam; Briggs, Michael E.; Stephenson, Andrew; Thomas, K. M.; Armstrong, Jayne A.; Bell, Jon; Busto, Jose; Noel, Raymond; Liu, Jian; Strachan, Denis M.; Thallapally, Praveen K.; Cooper, Andrew I.

    2014-10-31

    Abstract: The rare gases krypton, xenon, and radon pose both an economic opportunity and a potential environmental hazard. Xenon is used in commercial lighting, medical imaging, and anesthesia, and can sell for $5,000 per kilogram. Radon, by contrast, Is naturally radioactive and the second largest cause of lung cancer, and radioactive xenon, 133Xe, was a major pollutant released In the Fukushima Daiichi Nuclear Power Plant disaster. We describe an organic cage molecule that can capture xenon and radon with unprecedented selectivity, suggesting new technologies for environmental monitoring, removal of pollutants, or the recovery of rare, valuable elements from air.

  7. Cross-Linked Hyperbranched Polyglycerols as Hosts for Selective Binding of Guest Molecules

    PubMed Central

    Burakowska, Ewelina; Quinn, Jordan R.; Zimmerman, Steven C.; Haag, Rainer

    2009-01-01

    The ring-closing metathesis reaction of dendrimers containing allyl ether end groups is known to rigidify them significantly. Herein we report that polyallylated hyperbranched polyglycerol (HPG) 1 complexes the sodium salt of rose Bengal in chloroform solution but releases it readily to water. In contrast, extensively cross-linking 1 with Grubbs catalyst provides 2 which similarly complexes rose Bengal, but does not release it despite 12 h of shaking with water. Both 1 and 2 also complex thymol blue and exhibit the same differential complex stability when extracted with water. Neither 1 nor 2 complex Congo red sodium salt and more weakly solubilize the cesium salt of rose Bengal and thymol blue. Larger loop size cross-linked analogs HPG 5 and 6 also bind rose Bengal (RB) and thymol blue and are able to bind Congo red, but both release the dye more readily when extracted with water. In addition, a bathochromic shift is observed in the UV spectra for complex 6·RB, suggesting a changed microenvironment for the dye due to a tighter binding of the counter anion. Dihydroxylation of the alkene groups in 1, 2, 5, and 6 produced HPGs 3, 4, 7, and 8, respectively. HPGs 3 and 4 are both water-soluble, but 7 and 8 were not and could not be studied further. In water, HPG 4 solubilized less than one nonpolar guest (Nimodipine, pyrene, or Nile red) per polymer at least in part because it forms very large aggregates. Dynamic light scattering (DLS) and size exclusion chromatography (SEC) indicate aggregates with diameters of ca. 100 nm in pure water. The aggregates dissociated in high salt concentrations suggesting applications in stimuli responsive materials. PMID:19722631

  8. Identification of a Fragment-like Small Molecule Ligand for the Methyl-lysine Binding Protein, 53BP1

    PubMed Central

    Perfetti, Michael T.; Baughma, Brandi M.; Dickson, Bradley M.; Mu, Yunxiang; Cui, Gaofeng; Mader, Pavel; Dong, Aiping; Norris, Jacqueline L.; Rothbart, Scott B.; Strahl, Brian D.; Brown, Peter J.; Janzen, William P.; Arrowsmith, Cheryl H.; Mer, Georges; McBride, Kevin M.

    2015-01-01

    Improving our understanding of the role of chromatin regulators in the initiation, development, and suppression of cancer and other devastating diseases is critical, as they are integral players in regulating DNA integrity and gene expression. Developing small molecule inhibitors for this target class with cellular activity is a crucial step toward elucidating their specific functions. We specifically targeted the DNA damage response protein, 53BP1, which uses its tandem tudor domain to recognize histone H4 dimethylated on lysine 20 (H4K20me2), a modification induced by double-strand DNA breaks. Through a cross-screening approach we identified UNC2170 (1) as a micromolar ligand of 53BP1, which demonstrates at least 17-fold selectivity for 53BP1 as compared to other methyl-lysine (Kme) binding proteins tested. Structural studies revealed that the tert-butyl amine of UNC2170 anchors the compound in the methyl-lysine (Kme) binding pocket of 53BP1, making it competitive with endogenous Kme substrates. X-ray crystallography also demonstrated that UNC2170 binds at the interface of two tudor domains of a 53BP1 dimer. Importantly, this compound functions as a 53BP1 antagonist in cellular lysates and shows cellular activity by suppressing class switch recombination, a process which requires a functional 53BP1 tudor domain. These results demonstrate that UNC2170 is a functionally active, fragment-like ligand for 53BP1. PMID:25590533

  9. Identification of a fragment-like small molecule ligand for the methyl-lysine binding protein, 53BP1.

    PubMed

    Perfetti, Michael T; Baughman, Brandi M; Dickson, Bradley M; Mu, Yunxiang; Cui, Gaofeng; Mader, Pavel; Dong, Aiping; Norris, Jacqueline L; Rothbart, Scott B; Strahl, Brian D; Brown, Peter J; Janzen, William P; Arrowsmith, Cheryl H; Mer, Georges; McBride, Kevin M; James, Lindsey I; Frye, Stephen V

    2015-04-17

    Improving our understanding of the role of chromatin regulators in the initiation, development, and suppression of cancer and other devastating diseases is critical, as they are integral players in regulating DNA integrity and gene expression. Developing small molecule inhibitors for this target class with cellular activity is a crucial step toward elucidating their specific functions. We specifically targeted the DNA damage response protein, 53BP1, which uses its tandem tudor domain to recognize histone H4 dimethylated on lysine 20 (H4K20me2), a modification related to double-strand DNA breaks. Through a cross-screening approach, we identified UNC2170 (1) as a micromolar ligand of 53BP1, which demonstrates at least 17-fold selectivity for 53BP1 as compared to other methyl-lysine (Kme) binding proteins tested. Structural studies revealed that the tert-butyl amine of UNC2170 anchors the compound in the methyl-lysine (Kme) binding pocket of 53BP1, making it competitive with endogenous Kme substrates. X-ray crystallography also demonstrated that UNC2170 binds at the interface of two tudor domains of a 53BP1 dimer. Importantly, this compound functions as a 53BP1 antagonist in cellular lysates and shows cellular activity by suppressing class switch recombination, a process which requires a functional 53BP1 tudor domain. These results demonstrate that UNC2170 is a functionally active, fragment-like ligand for 53BP1.

  10. Single-molecule imaging reveals the mechanism of Exo1 regulation by single-stranded DNA binding proteins

    PubMed Central

    Gallardo, Ignacio F.; Zhou, Yi; Gong, Fade; Yang, Soo-Hyun; Wold, Marc S.; Miller, Kyle M.; Paull, Tanya T.

    2016-01-01

    Exonuclease 1 (Exo1) is a 5′→3′ exonuclease and 5′-flap endonuclease that plays a critical role in multiple eukaryotic DNA repair pathways. Exo1 processing at DNA nicks and double-strand breaks creates long stretches of single-stranded DNA, which are rapidly bound by replication protein A (RPA) and other single-stranded DNA binding proteins (SSBs). Here, we use single-molecule fluorescence imaging and quantitative cell biology approaches to reveal the interplay between Exo1 and SSBs. Both human and yeast Exo1 are processive nucleases on their own. RPA rapidly strips Exo1 from DNA, and this activity is dependent on at least three RPA-encoded single-stranded DNA binding domains. Furthermore, we show that ablation of RPA in human cells increases Exo1 recruitment to damage sites. In contrast, the sensor of single-stranded DNA complex 1—a recently identified human SSB that promotes DNA resection during homologous recombination—supports processive resection by Exo1. Although RPA rapidly turns over Exo1, multiple cycles of nuclease rebinding at the same DNA site can still support limited DNA processing. These results reveal the role of single-stranded DNA binding proteins in controlling Exo1-catalyzed resection with implications for how Exo1 is regulated during DNA repair in eukaryotic cells. PMID:26884156

  11. Theoretical study of the binding energy of a methane molecule in a (H2O)20 dodecahedral cage.

    PubMed

    Deible, Michael J; Tuguldur, Odbadrakh; Jordan, Kenneth D

    2014-07-17

    The interaction energy of a methane molecule encapsulated in a dodecahedral water cage is calculated using the MP2, MP2C, various dispersion-corrected DFT, and diffusion Monte Carlo (DMC) methods. The MP2, MP2C, and DMC methods give binding energies of -5.04, -4.60, and -5.3 ± 0.5 kcal/mol, respectively. In addition, the two- and three-body contributions are evaluated using the DFT, MP2, and CCSD(T) methods. All of the DFT methods considered appreciably overestimate the magnitude of the three-body contribution to the interaction energy. The two- and three-body energies are further analyzed by use of symmetry-adapted perturbation theory (SAPT) which allows decomposition into electrostatics, exchange, induction, and dispersion contributions. The SAPT calculations reveal that the induction, dispersion, and exchange three-body contributions to the methane-cage binding energy are all sizable, with the net three-body contribution to the binding energy being about 1 kcal/mol.

  12. Zinc Induces Dimerization of the Class II Major Histocompatibility Complex Molecule That Leads to Cooperative Binding to a Superantigen

    SciTech Connect

    Li,H.; Zhao, Y.; Guo, Y.; Li, Z.; Eislele, L.; Mourad, W.

    2007-01-01

    Dimerization of class II major histocompatibility complex (MHC) plays an important role in the MHC biological function. Mycoplasma arthritidis-derived mitogen (MAM) is a superantigen that can activate large fractions of T cells bearing specific T cell receptor V{beta} elements. Here we have used structural, sedimentation, and surface plasmon resonance detection approaches to investigate the molecular interactions between MAM and the class II MHC molecule HLA-DR1 in the context of a hemagglutinin peptide-(306-318) (HA). Our results revealed that zinc ion can efficiently induce the dimerization of the HLA-DR1/HA complex. Because the crystal structure of the MAM/HLA-DR1/hemagglutinin complex in the presence of EDTA is nearly identical to the structure of the complex crystallized in the presence of zinc ion, Zn{sup 2+} is evidently not directly involved in the binding between MAM and HLA-DR1. Sedimentation and surface plasmon resonance studies further revealed that MAM binds the HLA-DR1/HA complex with high affinity in a 1:1 stoichiometry, in the absence of Zn{sup 2+}. However, in the presence of Zn{sup 2+}, a dimerized MAM/HLA-DR1/HA complex can arise through the Zn{sup 2+}-induced DR1 dimer. In the presence of Zn{sup 2+}, cooperative binding of MAM to the DR1 dimer was also observed.

  13. Nucleic acid-binding molecules with high affinity and base sequence specificity: intercalating agents covalently linked to oligodeoxynucleotides.

    PubMed Central

    Asseline, U; Delarue, M; Lancelot, G; Toulmé, F; Thuong, N T; Montenay-Garestier, T; Hélène, C

    1984-01-01

    Oligodeoxyribonucleotides covalently linked to an intercalating agent via a polymethylene linker were synthesized. Oligothymidylates attached to an acridine dye (Acr) through the 3'-phosphate group [(Tp)n(CH2) mAcr ] specifically interact with the complementary sequence. The interaction is strongly stabilized by the intercalating agent. By using absorption and fluorescence spectroscopies, it is shown that complex formation between (Tp)n(CH2) mAcr and poly(rA) involves the formation of n A X T base pairs, where n is the number of thymines in the oligonucleotide. The acridine ring intercalates between A X T base pairs. Fluorescence excitation spectra reveal the existence of two environments for the acridine ring, whose relative contributions depend on the linker length (m). The binding of (Tp)4(CH2) mAcr to poly(rA) is analyzed in terms of site binding and cooperative interactions between oligonucleotides along the polynucleotide lattice. Thermodynamic parameters show that the covalent attachment of the acridine ring strongly stabilizes the binding of the oligonucleotide to its complementary sequence. The stabilization depends on the linker length; the compound with m = 5 gives a more stable complex than that with m = 3. These results open the way to the synthesis of a family of molecules exhibiting both high-affinity and high-specificity for a nucleic acid base sequence. PMID:6587350

  14. Improved Estimation of Protein-Ligand Binding Free Energy by Using the Ligand-Entropy and Mobility of Water Molecules

    PubMed Central

    Fukunishi, Yoshifumi; Nakamura, Haruki

    2013-01-01

    We previously developed the direct interaction approximation (DIA) method to estimate the protein-ligand binding free energy (ΔG). The DIA method estimates the ΔG value based on the direct van der Waals and electrostatic interaction energies between the protein and the ligand. In the current study, the effect of the entropy of the ligand was introduced with protein dynamic properties by molecular dynamics simulations, and the interaction between each residue of the protein and the ligand was also weighted considering the hydration of each residue. The molecular dynamics simulation of the apo target protein gave the hydration effect of each residue, under the assumption that the residues, which strongly bind the water molecules, are important in the protein-ligand binding. These two effects improved the reliability of the DIA method. In fact, the parameters used in the DIA became independent of the target protein. The averaged error of ΔG estimation was 1.3 kcal/mol and the correlation coefficient between the experimental ΔG value and the calculated ΔG value was 0.75. PMID:24276169

  15. Zinc Induces Dimerization of the Class II Major Histocompatibility Complex Molecule That Leads to Cooperative Binding to a Superantigen*

    PubMed Central

    Li, Hongmin; Zhao, Yiwei; Guo, Yi; Li, Zhong; Eisele, Leslie; Mourad, Walid

    2014-01-01

    Dimerization of class II major histocompatibility complex (MHC) plays an important role in the MHC biological function. Mycoplasma arthritidis-derived mitogen (MAM) is a superantigen that can activate large fractions of T cells bearing specific T cell receptor Vβ elements. Here we have used structural, sedimentation, and surface plasmon resonance detection approaches to investigate the molecular interactions between MAM and the class II MHC molecule HLA-DR1 in the context of a hemagglutinin peptide-(306–318) (HA). Our results revealed that zinc ion can efficiently induce the dimerization of the HLA-DR1/HA complex. Because the crystal structure of the MAM/HLA-DR1/hemagglutinin complex in the presence of EDTA is nearly identical to the structure of the complex crystallized in the presence of zinc ion, Zn2+ is evidently not directly involved in the binding between MAM and HLA-DR1. Sedimentation and surface plasmon resonance studies further revealed that MAM binds the HLA-DR1/HA complex with high affinity in a 1:1 stoichiometry, in the absence of Zn2+. However, in the presence of Zn2+, a dimerized MAM/HLA-DR1/HA complex can arise through the Zn2+-induced DR1 dimer. In the presence of Zn2+, cooperative binding of MAM to the DR1 dimer was also observed. PMID:17166841

  16. A TonB-dependent outer membrane protein as a Bacteroides fragilis fibronectin-binding molecule.

    PubMed

    Pauer, Heidi; Ferreira, Eliane de Oliveira; dos Santos-Filho, Joaquim; Portela, Maristela Barbosa; Zingali, Russolina Benedeta; Soares, Rosangela Maria Araújo; Domingues, Regina Maria Cavalcanti Pilotto

    2009-04-01

    The binding of Bacteroides fragilis to plasmatic fibronectin was investigated using strains isolated from healthy subjects and from patients with bacteremia. They were cultivated in a synthetic media in which variations in cysteine concentrations determined alterations in the oxidation-reduction potential (Eh). All the strains assayed were capable of adhering to plasmatic fibronectin when cultivated under oxidizing and reducing conditions. Bacteroides fragilis 1405 showed the greatest difference when the results under these conditions were compared and it was selected for further investigations. Chemical treatments suggested the involvement of a protein in the interaction between B. fragilis and plasmatic fibronectin. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis of outer membrane proteins (OMPs) revealed differences between the extracts obtained from cultures grown under the two conditions. Protein bands of c. 102, 100, 77, 73, 50 and 40 kDa were more highly expressed under oxidizing than reducing conditions. Dot blot analysis showed a stronger recognition of plasmatic fibronectin by OMPs obtained from cultures grown under higher Eh, and Western blot assays confirmed a band of c. 102 kDa as fibronectin-binding protein. This protein was sequenced and revealed to be a putative TonB-dependent OMPs. PCR analysis confirmed the presence of this gene in all the studied strains.

  17. Development of an efficient G-quadruplex stabilized thrombin binding aptamer containing 3-carbon spacer molecule.

    PubMed

    Naduvile Veedu, Rakesh; Aaldering, Lukas; Poongavanam, Vasanthanathan; Langkjaer, Niels; Murugan, Arul; Jørgensen, Per Trolle; Wengel, Jesper

    2017-02-02

    Thrombin binding aptamer (TBA) that shows anticoagulant properties is one of the most studied G-quadruplex forming aptamers. In this study, we investigated the impact of different chemical modifications such as Spacer-C3, unlocked nucleic acid (UNA) and 3'-amine-modified UNA (amino-UNA) on the structural dynamics and stability of TBA. All three modifications were incorporated at three different loop positions (T3, T7 and T12) of the TBA G-quadruplex structure rendering a series of TBA variants and studied their stability by thermal denaturation studies, folding by circular dichroism spectroscopy and thrombin clotting time. The results showed that Spacer-C3 introduction at T7 loop position (TBA-SP7) significantly improved the stability and thrombin clotting time while maintaining a similar binding affinity as TBA to thrombin. Detailed molecular modelling experiments provided novel insights to the experimental observation that further supported the efficacy of TBA-SP7. The results of this study could provide valuable information for future designs of TBA analogues with superior thrombin inhibition properties.

  18. γ Sulphate PNA (PNA S): highly selective DNA binding molecule showing promising antigene activity.

    PubMed

    Avitabile, Concetta; Moggio, Loredana; Malgieri, Gaetano; Capasso, Domenica; Di Gaetano, Sonia; Saviano, Michele; Pedone, Carlo; Romanelli, Alessandra

    2012-01-01

    Peptide Nucleic Acids (PNAs), nucleic acid analogues showing high stability to enzyme degradation and strong affinity and specificity of binding toward DNA and RNA are widely investigated as tools to interfere in gene expression. Several studies have been focused on PNA analogues with modifications on the backbone and bases in the attempt to overcome solubility, uptake and aggregation issues. γ PNAs, PNA derivatives having a substituent in the γ position of the backbone show interesting properties in terms of secondary structure and affinity of binding toward complementary nucleic acids. In this paper we illustrate our results obtained on new analogues, bearing a sulphate in the γ position of the backbone, developed to be more DNA-like in terms of polarity and charge. The synthesis of monomers and oligomers is described. NMR studies on the conformational properties of monomers and studies on the secondary structure of single strands and triplexes are reported. Furthermore the hybrid stability and the effect of mismatches on the stability have also been investigated. Finally, the ability of the new analogue to work as antigene, interfering with the transcription of the ErbB2 gene on a human cell line overexpressing ErbB2 (SKBR3), assessed by FACS and qPCR, is described.

  19. Old and new findings on lipopolysaccharide-binding protein: a soluble pattern-recognition molecule.

    PubMed

    Schumann, Ralf R

    2011-08-01

    LBP [LPS (lipopolysaccharide)-binding protein] was discovered approximately 25 years ago. Since then, substantial progress has been made towards our understanding of its function in health and disease. Furthermore, the discovery of a large protein family sharing functional and structural attributes has helped in our knowledge. Still, key questions are unresolved, and here an overview on the old and new findings on LBP is given. LBP is an acute-phase protein of the liver, but is also synthesized in other cells of the organism. While LBP is named after the ability to bind to LPS of Gram-negative bacteria, it also can recognize other bacterial compounds, such as lipopeptides. It has been shown that LBP is needed to combat infections; however, the main mechanism of action is still not clear. New findings on natural genetic variations of LBP leading to functional consequences may help in further elucidating the mechanism of LBP and its role in innate immunity and disease.

  20. Hydrogen molecule binding to unsaturated metal sites in metal-organic frameworks studied by neutron powder diffraction and inelastic neutron scattering

    NASA Astrophysics Data System (ADS)

    Liu, Yun; Brown, Craig; Neumann, Dan; Dinca, Mircea; Long, Jeffrey; Peterson, Vanessa; Kepert, Cameron

    2007-03-01

    Metal organic framework (MOF) materials have shown considerable potential for hydrogen storage arising from very large surface areas. However, the low binding energy of hydrogen molecules limits its storage capability to very low temperatures (< 77 K), which is impractical for industrial applications. Using neutron powder diffraction (NPD), we have characterized the hydrogen adsorption sites in a selected series of MOF materials with exposed unsaturated metal ions. Direct binding between the unsaturated metal ions and hydrogen molecules is observed and responsible for the enhanced initial hydrogen adsorption enthalpy. The different metals centers in these MOFs show different binding strength and interaction distances between the hydrogen molecule and metal ions. The organic linker also affects the overall H2 binding strength. Inelastic neutron scattering spectra of H2 in these MOFs are also discussed.

  1. Experimental and theoretical anion binding studies on coumarin linked thiourea and urea molecules

    NASA Astrophysics Data System (ADS)

    Ghosh, Kumaresh; Adhikari, Suman; Fröhlich, Roland; Petsalakis, Ioannis D.; Theodorakopoulos, Giannoula

    2011-10-01

    A series of coumarin linked thiourea and urea molecules 1- 5 have been designed and synthesized. Thiourea-based monotopic receptors 1- 4 showed selective sensing of F - and C 6H 5COO - by exhibiting significant change in emission as well as change in color. The ditopic receptor 5, on the other hand, fluorometrically distinguished isomeric aromatic dicarboxylates in polar solvent DMSO. Theoretical calculations have been carried out on systems 1-4 and their complexes with C 6H 5COO - and F - anions in the presence of CH 3CN solvent. Calculations have been carried out to determine the geometry and bonding of the complexes in the ground electronic state as well as on the excited electronic states relevant to the absorption spectra and emission spectra in an effort to obtain information on the spectral changes accompanying complexation of the receptors 1-4 with the two anions.

  2. [Calculation of mobility and entropy of the binding of molecules by crystals].

    PubMed

    Garbuzynskiy, S O; Finkelstein, A V

    2016-01-01

    A simple method for evaluating a range of molecular movements in crystals has been developed. This estimate is needed to calculate the entropy of binding, in particular in protein-ligand complexes. The estimate is based on experimental data concerning the enthalpy of sublimation and saturated vapor pressure obtained for 15 organic crystals with melting temperatures of 25-80°С. For this set, we calculated the values of the average range and the corresponding average amplitude of molecular movements in crystals that constituted 0.75 ± 0.14 Å and 0.18 ± 0.03 Å, respectively. The entropy of sublimation calculated based on the average range of molecular movements in crystals was well consistent with the experimental data.

  3. More powerful virus inhibitors from structure-based analysis of HEV71 capsid-binding molecules

    PubMed Central

    Spyrou, John A. B.; Kelly, James; Ren, Jingshan; Grimes, Jonathan; Puerstinger, Gerhard; Stonehouse, Nicola; Walter, Thomas S.; Hu, Zhongyu; Wang, Junzhi; Li, Xuemei; Peng, Wei; Rowlands, David; Fry, Elizabeth E.; Rao, Zihe; Stuart, David I.

    2014-01-01

    Enterovirus 71 (HEV71) epidemics amongst children and infants result mainly in mild symptoms, however, especially in the Asia-Pacific region, infection can be fatal. At present no therapies are available. We have used structural analysis of the complete virus to guide the design of HEV71 inhibitors. Analysis of complexes with four 3-(-4-pyridyl)-2-imidazolidinone derivatives with varying anti-HEV71 activities, pinpointed key structure-activity correlates. We then identified additional potentially beneficial substitutions, developed methods to reliably triage compounds by quantum mechanics-enhanced ligand docking, and synthesized two candidates. Structural analysis and in vitro assays confirmed the predicted binding modes and their ability to block viral infection. One ligand (IC50 = 25 pM) is an order of magnitude more potent than the best previously reported inhibitor, and is also more soluble. Our approach may be useful in the design of effective drugs for enterovirus infections. PMID:24509833

  4. An overview of the binding models of FGFR tyrosine kinases in complex with small molecule inhibitors.

    PubMed

    Cheng, Weiyan; Wang, Mixiang; Tian, Xin; Zhang, Xiaojian

    2017-01-27

    The fibroblast growth factor receptor (FGFR) family receptor tyrosine kinase (RTK) includes four structurally related members, termed as FGFR1, FGFR2, FGFR3, and FGFR4. Given its intimate role in the progression of several solid tumors, excessive FGFR signaling provides an opportunity for anticancer therapy. Along with extensive pharmacological studies validating the therapeutic potential of targeting the FGFRs for cancer treatment, co-crystal structures of FGFRs/inhibitors are continuously coming up to study the mechanism of actions and explore new inhibitors. Herein, we review the reported co-crystals of FGFRs in complex with the corresponding inhibitors, main focusing our attention on the binding models and the pharmacological activities of the inhibitors.

  5. Probing Binding and Cellular Activity of Pyrrolidinone and Piperidinone Small Molecules Targeting the Urokinase Receptor

    PubMed Central

    Mani, Timmy; Liu, Degang; Zhou, Donghui; Li, Liwei; Knabe, William Eric; Wang, Fang; Oh, Kyungsoo; Meroueh, Samy O.

    2014-01-01

    The urokinase receptor (uPAR) is a cell-surface protein that is part of an intricate web of transient and tight protein interactions that promote cancer cell invasion and metastasis. Here we evaluate the binding and biological activity of a new class of pyrrolidinone (3) and piperidinone (4) compounds, along with derivatives of previously-identified pyrazole (1) and propylamine (2) compounds. Competition assays revealed that the compounds displaced a fluorescently-labeled peptide (AE147-FAM) with inhibition constant Ki ranging from 6 to 63 μM. Structure-based computational pharmacophore analysis followed by extensive explicit-solvent molecular dynamics simulations and free energy calculations suggested pyrazole-based 1a and piperidinone-based 4 adopt different binding modes, despite their similar two-dimensional structures. In cells, compounds 1b and 1f showed significant inhibition of breast MDA-MB-231 and pancreatic ductal adenocarcinoma (PDAC) cell proliferation, but 4b exhibited no cytotoxicity even at concentrations of 100 μM. 1f impaired MDA-MB-231 invasion, adhesion, and migration in a concentration-dependent manner, while 4b inhibited only invasion. 1f inhibited gelatinase (MMP-9) activity in a concentration-dependent manner, while 4b showed no effect suggesting different mechanisms for inhibition of cell invasion. Signaling studies further highlighted these differences, showing that pyrazole compounds completely inhibited ERK phosphorylation and impaired HIF1α and NF-κB signaling, while pyrrolidinone and piperidinone (3 and 4b) had no effect. Annexin V staining suggested that the effect of pyrazole-based 1f on proliferation was due to cell killing through an apoptotic mechanism. PMID:24115356

  6. Interaction of small molecules with double-stranded RNA: spectroscopic, viscometric, and calorimetric study of hoechst and proflavine binding to PolyCG structures.

    PubMed

    Sinha, Rangana; Hossain, Maidul; Kumar, Gopinatha Suresh

    2009-04-01

    Design and synthesis of new small molecules binding to double-stranded RNA necessitate complete understanding of the molecular aspects of the binding of many existing molecules. Toward this goal, in this work we evaluated the biophysical aspects of the interaction of a DNA intercalator (proflavine) and a minor groove binder (hoechst 33258) with two polymorphic forms of polyCG, namely, the right-handed Watson-Crick base paired A-form and the left-handed Hoogsteen base paired H(L)-form, by absorption, fluorescence, and viscometry experiments. The energetics of the interaction of these molecules with the RNA structures has also been elucidated by isothermal titration calorimetry (ITC). Results suggest that proflavine strongly intercalates in both forms of polyCG, whereas hoechst shows mainly groove-binding modes. The binding of both drugs to both forms of RNA resulted in significant conformational change to the RNA structure with the bound molecules being placed in the chiral RNA helix. ITC profiles for both proflavine and hoechst show two binding sites. Binding of proflavine to both forms of RNA is endothermic and entropy driven in the first site and exothermic and enthalpy driven in the second site, whereas hoechst binding to both forms of RNA is exothermic and enthalpy driven in the first site and endothermic and entropy driven in the second site. This study suggests that the binding affinity characteristics and energetics of interaction of these DNA binding molecules with the RNA conformations are significantly different and may serve as data for future development of effective structure-selective RNA-based drugs.

  7. Photoelectron spectroscopy of s-triazine anion clusters: Polarization-induced electron binding in aza-aromatic molecule

    NASA Astrophysics Data System (ADS)

    Kim, Jeong Hyun; Song, Jae Kyu; Park, Hyokeun; Lee, Sang Hak; Han, Sang Yun; Kim, Seong Keun

    2003-08-01

    Photoelectron spectroscopy was carried out for the mass-selected cluster anions of s-triazine molecule, Tzn- (n=1-6). The mass spectrum and vibrationally resolved photoelectron spectrum of Tz- showed that unlike pyridine and pyrazine, Tz binds an electron and thus becomes the first molecule in the azabenzene series with a positive electron affinity (0.03 eV). This indicates that the local charge polarization in the aromatic ring by the three nitrogen atoms is large enough to facilitate electron binding to a homologue of benzene. A Jahn-Teller distortion was proposed to explain the vibrational progressions of the photoelectron spectrum of Tz-. A series of Ar-solvated clusters of Tz-, Tz-ṡArm (m=1-7), have been also studied. Their photoelectron spectra showed a drop in the incremental electron binding energy when going from m=4 to 5, indicating the closure of a solvation shell with four Ar atoms. In the mass abundance spectrum of Tzn-, a distinctly high intensity for Tz2- indicated its exceptional stability, which was also manifested by the large increase by more than 0.5 eV in the vertical detachment energy of the photoelectron spectrum. Theoretical calculations were carried out to obtain optimized geometries of the neutral and anion of Tz and Tz2. We confirmed the Jahn-Teller distortion in Tz- and also addressed the role of hydrogen bonding in determining the geometries of Tz2-. A common feature for the two most stable forms of Tz2- with comparable energies was that they achieve their unique stability through equal sharing of the negative charge between their two molecular constituents. A new photoelectron band was found to emerge from Tzn- for n⩾2 by the 355 nm light, in addition to the photoelectron band at lower electron binding energy observed for n⩾1 at 532 nm. The relative intensity of this new band decreased as n increased, and its position was 1.6-1.8 eV above the first band. Photodetachment to an electronically excited state was suggested to give

  8. Freezing adhesion molecules in a state of high-avidity binding blocks eosinophil migration

    PubMed Central

    1993-01-01

    Leukocyte extravasation is mediated by multiple interactions of adhesive surface structures with ligands on endothelial cells and matrix components. The functional role of beta 1 (CD29) integrins (or very late antigen [VLA] proteins) in eosinophil migration across polycarbonate filters was examined under several in vitro conditions. Eosinophil migration induced by the chemoattractant C5a or platelet- activating factor was fully inhibited by monoclonal antibody (mAb) 8A2, a recently characterized "activating" CD29 mAb. However, inhibition by mAb 8A2 was observed only under filter conditions that best reflected the in vivo situation, i.e., when the eosinophils migrated over filters preincubated with the extracellular matrix (ECM) protein fibronectin (FN), or when the filters were covered with confluent monolayers of cultured human umbilical vein endothelial cells (HUVEC). When bare untreated filters were used, mAb 8A2 had no effect, whereas the C5a- directed movement was prevented by CD18 mAb. Studies with alpha-subunit (CD49)-specific mAbs indicated that the integrins VLA-4 and -5 mediated migration across FN-preincubated filters, and VLA-2, -4, -5, and -6 were involved in eosinophil migration through filters covered with HUVEC. In contrast with the activating CD29 mAb 8A2, a combination of blocking CD49 mAbs or the nonactivating but blocking CD29 mAb AIIB2 failed to inhibit completely eosinophil migration over FN-preincubated or HUVEC-covered filters. mAb 8A2 stimulated binding to FN but not to HUVEC. Moreover, eosinophil migration over FN-preincubated or HUVEC- covered filters was significantly inhibited by anti-connecting segment 1 (CS-1) mAbs, as well as the soluble CS-1 peptide (unlike migration across bare untreated filters). Thus, inhibition of eosinophil migration by mAb 8A2 depended upon the presence of ECM proteins and not upon the presence of HUVEC per se. In conclusion, "freezing" adhesion receptors of the beta 1 integrin family into their high

  9. Targeting Human Gastrointestinal Stromal Tumour Cells with a Quadruplex-binding Small Molecule

    PubMed Central

    Gunaratnam, Mekala; Beltran, Monica; Galesa, Katja; Haider, Shozeb M.; Reszka, Anthony P.; Cuenca, Francisco; Fletcher, Jonathan A.; Neidle, Stephen

    2010-01-01

    The majority of human gastrointestinal stromal tumours (GIST) are driven by activating mutations in the proto-oncogene KIT, a tyrosine kinase receptor. Clinical treatment with imatinib targets the kinase domain of KIT, but tumour regrowth occurs as a result of the development of resistant mutations in the kinase active site. An alternative small-molecule approach to GIST therapy is described, in which the KIT gene is directly targeted, and thus kinase resistance may be circumvented. A naphthalene dimiide derivative has been used to demonstrate the concept of dual quadruplex targeting. This compound strongly stabilises both telomeric quadruplex DNA and quadruplex sites in the KIT promoter in vitro. It is shown here that the compound is a potent inducer of growth arrest in a patient-derived GIST cell line at a concentration (ca 1μM) that also results in effective inhibition of telomerase activity and almost complete suppression of KIT mRNA and KIT protein expression. Molecular modelling studies with a telomeric quadruplex have been used to rationalise aspects of the experimental quadruplex melting data. PMID:19469547

  10. Assessment of two hybrid van der Waals density functionals for covalent and non-covalent binding of molecules

    NASA Astrophysics Data System (ADS)

    Berland, Kristian; Jiao, Yang; Lee, Jung-Hoon; Rangel, Tonatiuh; Neaton, Jeffrey B.; Hyldgaard, Per

    2017-06-01

    Two hybrid van der Waals density functionals (vdW-DFs) are developed using 25% Fock exchange with (i) the consistent-exchange vdW-DF-cx functional [K. Berland and P. Hyldgaard, Phys. Rev. B 89, 035412 (2014)] and (ii) with the vdW-DF2 functional [K. Lee et al., Phys. Rev. B 82, 081101 (2010)]. The ability to describe covalent and non-covalent binding properties of molecules is assessed. For properties related to covalent binding, atomization energies (G2-1 set), molecular reaction energies (G2RC set), and ionization energies (G21IP set) are benchmarked against experimental reference values. We find that hybrid-vdW-DF-cx yields results that are rather similar to those of the standard non-empirical hybrid PBE0 [C. Adamo and V. Barone, J. Chem. Phys. 110, 6158 (1999)], with mean average deviations (MADs) of 4.9 and 5.0 kcal/mol for the G2-1 set, respectively. In this comparison, experimental reference values are used, back corrected by wavefunction-based quantum-chemistry calculations of zero-point energies. Hybrid vdW-DF2 follows somewhat different trends, showing on average significantly larger deviations from the reference energies, with a MAD of 14.5 kcal/mol for the G2-1 set. Non-covalent binding properties of molecules are assessed using the S22 benchmark set of non-covalently bonded dimers and the X40 set of dimers of small halogenated molecules, using wavefunction-based quantum chemistry results as references. For the S22 set, hybrid-vdW-DF-cx performs better than standard vdW-DF-cx for the mostly hydrogen-bonded systems, with MAD dropping from 0.6 to 0.3 kcal/mol, but worse for purely dispersion-bonded systems, with MAD increasing from 0.2 to 0.6 kcal/mol. Hybrid-vdW-DF2 offers a slight improvement over standard vdW-DF2. Similar trends are found for the X40 set, with hybrid-vdW-DF-cx performing particularly well for binding energies involving the strongly polar hydrogen halides, but poorly for systems with tiny binding energies. Our study of the X40 set

  11. In silico derived small molecules bind the filovirus VP35 protein and inhibit its polymerase co-factor activity

    PubMed Central

    Brown, Craig S.; Lee, Michael S.; Leung, Daisy W.; Wang, Tianjiao; Xu, Wei; Luthra, Priya; Anantpadma, Manu; Shabman, Reed S.; Melito, Lisa M.; MacMillan, Karen S.; Borek, Dominika M.; Otwinowski, Zbyszek; Ramanan, Parameshwaran; Stubbs, Alisha J.; Peterson, Dayna S.; Binning, Jennifer M.; Tonelli, Marco; Olson, Mark A.; Davey, Rob; Ready, Joseph M.; Basler, Christopher F.; Amarasinghe, Gaya K.

    2014-01-01

    The Ebola virus (EBOV) genome only encodes a single viral polypeptide with enzymatic activity, the viral Large (L) RNA-dependent RNA polymerase protein. However, currently there is limited information about L protein, which has hampered development of antivirals. Therefore, antifiloviral therapeutic efforts must include additional targets such as protein-protein interfaces (PPIs). Viral protein 35 (VP35) is multifunctional and plays important roles in viral pathogenesis, including viral mRNA synthesis and replication of the negative-sense RNA viral genome. Previous studies revealed that mutation of key basic residues within the VP35 interferon inhibitory domain (IID) results in significant EBOV attenuation, both in vitro and in vivo. In the current study, we use an experimental pipeline that includes structure-based in silico screening, biochemical and structural characterization, along with medicinal chemistry to identify and characterize small molecules that target a binding pocket within VP35. NMR mapping experiments and high resolution x-ray crystal structures show that select small molecules bind to a region of VP35 IID that is important for replication complex formation through interactions with the viral nucleoprotein (NP). We also tested select compounds for their ability to inhibit VP35 IID-NP interactions in vitro as well as VP35 function in a minigenome assay and EBOV replication. These results confirm the ability of compounds identified in this study to inhibit VP35-NP interactions in vitro and to impair viral replication in cell-based assays. These studies provide an initial framework to guide development of antifiloviral compounds against filoviral VP35 proteins. PMID:24495995

  12. Early events of fertilization in sea urchin eggs are sensitive to actin-binding organic molecules.

    PubMed

    Chun, Jong T; Limatola, Nunzia; Vasilev, Filip; Santella, Luigia

    2014-08-01

    We previously demonstrated that many aspects of the intracellular Ca(2+) increase in fertilized eggs of starfish are significantly influenced by the state of the actin cytoskeleton. In addition, the actin cytoskeleton appeared to play comprehensive roles in modulating cortical granules exocytosis and sperm entry during the early phase of fertilization. In the present communication, we have extended our work to sea urchin which is believed to have bifurcated from the common ancestor in the phylogenetic tree some 500 million years ago. To corroborate our earlier findings in starfish, we have tested how the early events of fertilization in sea urchin eggs are influenced by four different actin-binding drugs that promote either depolymerization or stabilization of actin filaments. We found that all the actin drugs commonly blocked sperm entry in high doses and significantly reduced the speed of the Ca(2+) wave. At low doses, however, cytochalasin B and phalloidin increased the rate of polyspermy. Overall, certain aspects of Ca(2+) signaling in these eggs were in line with the morphological changes induced by the actin drugs. That is, the time interval between the cortical flash and the first Ca(2+) spot at the sperm interaction site (the latent period) was significantly prolonged in the eggs pretreated with cytochalasin B or latrunculin A, whereas the Ca(2+) decay kinetics after the peak was specifically attenuated in the eggs pretreated with jasplakinolide or phalloidin. In addition, the sperm interacting with the eggs pretreated with actin drugs often generated multiple Ca(2+) waves, but tended to fail to enter the egg. Thus, our results indicated that generation of massive Ca(2+) waves is neither indicative of sperm entry nor sufficient for cortical granules exocytosis in the inseminated sea urchin eggs, whereas the structure and functionality of the actin cytoskeleton are the major determining factors in the two processes. Copyright © 2014 Elsevier Inc. All rights

  13. Systematic Approach to the Development of Microfabricated Biosensors: Relationship between Gold Surface Pretreatment and Thiolated Molecule Binding.

    PubMed

    Makaraviciute, Asta; Xu, Xingxing; Nyholm, Leif; Zhang, Zhen

    2017-08-09

    Despite the increasing popularity of microfabricated biosensors due to advances in technologic and surface functionalization strategies, their successful implementation is partially inhibited by the lack of consistency in their analytical characteristics. One of the main causes for the discrepancies is the absence of a systematic and comprehensive approach to surface functionalization. In this article microfabricated gold electrodes aimed at biosensor development have been systematically characterized in terms of surface pretreatment, thiolated molecule binding, and reproducibility by means of X-ray photoelectron scattering (XPS) and cyclic voltammetry (CV). It has been shown that after SU-8 photolithography gold surfaces were markedly contaminated, which decreased the effective surface area and surface coverage of a model molecule mercaptohexanol (MCH). Three surface pretreatment methods compatible with microfabricated devices were compared. The investigated methods were (i) cyclic voltammetry in dilute H2SO4, (ii) gentle basic piranha followed by linear sweep voltammetry in dilute KOH, and (iii) oxygen plasma treatment followed by incubation in ethanol. It was shown that all three methods significantly decreased the contamination and increased MCH surface coverage. Most importantly, it was also revealed that surface pretreatments may induce structural changes to the gold surfaces. Accordingly, these alterations influence the characteristics of MCH functionalization.

  14. Structure of a small-molecule inhibitor complexed with GlmU from Haemophilus influenzae reveals an allosteric binding site

    SciTech Connect

    Mochalkin, Igor; Lightle, Sandra; Narasimhan, Lakshmi; Bornemeier, Dirk; Melnick, Michael; VanderRoest, Steven; McDowell, Laura

    2008-04-02

    N-Acetylglucosamine-1-phosphate uridyltransferase (GlmU) is an essential enzyme in aminosugars metabolism and an attractive target for antibiotic drug discovery. GlmU catalyzes the formation of uridine-diphospho-N-acetylglucosamine (UDP-GlcNAc), an important precursor in the peptidoglycan and lipopolisaccharide biosynthesis in both Gram-negative and Gram-positive bacteria. Here we disclose a 1.9 {angstrom} resolution crystal structure of a synthetic small-molecule inhibitor of GlmU from Haemophilus influenzae (hiGlmU). The compound was identified through a high-throughput screening (HTS) configured to detect inhibitors that target the uridyltransferase active site of hiGlmU. The original HTS hit exhibited a modest micromolar potency (IC{sub 50} - 18 {mu}M in a racemic mixture) against hiGlmU and no activity against Staphylococcus aureus GlmU (saGlmU). The determined crystal structure indicated that the inhibitor occupies an allosteric site adjacent to the GlcNAc-1-P substrate-binding region. Analysis of the mechanistic model of the uridyltransferase reaction suggests that the binding of this allosteric inhibitor prevents structural rearrangements that are required for the enzymatic reaction, thus providing a basis for structure-guided design of a new class of mechanism-based inhibitors of GlmU.

  15. Recruitment of RNA molecules by connexin RNA-binding motifs: Implication in RNA and DNA transport through microvesicles and exosomes.

    PubMed

    Varela-Eirin, Marta; Varela-Vazquez, Adrian; Rodríguez-Candela Mateos, Marina; Vila-Sanjurjo, Anton; Fonseca, Eduardo; Mascareñas, José L; Eugenio Vázquez, M; Mayan, Maria D

    2017-04-01

    Connexins (Cxs) are integral membrane proteins that form high-conductance plasma membrane channels, allowing communication from cell to cell (via gap junctions) and from cells to the extracellular environment (via hemichannels). Initially described for their role in joining excitable cells (nerve and muscle), gap junctions (GJs) are found between virtually all cells in solid tissues and are essential for functional coordination by enabling the direct transfer of small signalling molecules, metabolites, ions, and electrical signals from cell to cell. Several studies have revealed diverse channel-independent functions of Cxs, which include the control of cell growth and tumourigenicity. Connexin43 (Cx43) is the most widespread Cx in the human body. The myriad roles of Cx43 and its implication in the development of disorders such as cancer, inflammation, osteoarthritis and Alzheimer's disease have given rise to many novel questions. Several RNA- and DNA-binding motifs were predicted in the Cx43 and Cx26 sequences using different computational methods. This review provides insights into new, ground-breaking functions of Cxs, highlighting important areas for future work such as transfer of genetic information through extracellular vesicles. We discuss the implication of potential RNA- and DNA-binding domains in the Cx43 and Cx26 sequences in the cellular communication and control of signalling pathways.

  16. Discovery of inhibitors of aberrant gene transcription from Libraries of DNA binding molecules: inhibition of LEF-1-mediated gene transcription and oncogenic transformation.

    PubMed

    Stover, James S; Shi, Jin; Jin, Wei; Vogt, Peter K; Boger, Dale L

    2009-03-11

    The screening of a >9000 compound library of synthetic DNA binding molecules for selective binding to the consensus sequence of the transcription factor LEF-1 followed by assessment of the candidate compounds in a series of assays that characterized functional activity (disruption of DNA-LEF-1 binding) at the intended target and site (inhibition of intracellular LEF-1-mediated gene transcription) resulting in a desired phenotypic cellular change (inhibit LEF-1-driven cell transformation) provided two lead compounds: lefmycin-1 and lefmycin-2. The sequence of screens defining the approach assures that activity in the final functional assay may be directly related to the inhibition of gene transcription and DNA binding properties of the identified molecules. Central to the implementation of this generalized approach to the discovery of DNA binding small molecule inhibitors of gene transcription was (1) the use of a technically nondemanding fluorescent intercalator displacement (FID) assay for initial assessment of the DNA binding affinity and selectivity of a library of compounds for any sequence of interest, and (2) the technology used to prepare a sufficiently large library of DNA binding compounds.

  17. Discovery of Inhibitors of Aberrant Gene Transcription from Libraries of DNA Binding Molecules: Inhibition of LEF-1 Mediated Gene Transcription and Oncogenic Transformation

    PubMed Central

    Stover, James S.; Shi, Jin; Jin, Wei; Vogt, Peter K.; Boger, Dale L.

    2009-01-01

    The screening of a >9000 compound library of synthetic DNA binding molecules for selective binding to the consensus sequence of the transcription factor LEF-1 followed by assessment of the candidate compounds in a series of assays that characterized functional activity (disruption of DNA–LEF-1 binding) at the intended target and site (inhibition of intracellular LEF-1 mediated gene transcription) resulting in a desired phenotypic cellular change (inhibit LEF-1 driven cell transformation) provided two lead compounds: lefmycin-1 and lefmycin-2. The sequence of screens defining the approach assures that activity in the final functional assay may be directly related to the inhibition of gene transcription and DNA binding properties of the identified molecules. Central to the implementation of this generalized approach to the discovery of DNA binding small molecule inhibitors of gene transcription was: (1) the use of a technically non-demanding fluorescent intercalator displacement (FID) assay for initial assessment of the DNA binding affinity and selectivity of a library of compounds for any sequence of interest, and (2) the technology used to prepare a sufficiently large library of DNA binding compounds. PMID:19216569

  18. Effector-repressor interactions, binding of a single effector molecule to the operator-bound TtgR homodimer mediates derepression.

    PubMed

    Terán, Wilson; Krell, Tino; Ramos, Juan Luis; Gallegos, María-Trinidad

    2006-03-17

    The RND family transporter TtgABC and its cognate repressor TtgR from Pseudomonas putida DOT-T1E were both shown to possess multidrug recognition properties. Structurally unrelated molecules such as chloramphenicol, butyl paraben, 1,3-dihydroxynaphthalene, and several flavonoids are substrates of TtgABC and activate pump expression by binding to the TtgR-operator complex. Isothermal titration calorimetry was employed to determine the thermodynamic parameters for the binding of these molecules to TtgR. Dissociation constants were in the range from 1 to 150 microm, the binding stoichiometry was one effector molecule per dimer of TtgR, and the process was driven by favorable enthalpy changes. Although TtgR exhibits a large multidrug binding profile, the plant-derived compounds phloretin and quercetin were shown to bind with the highest affinity (K(D) of around 1 microm), in contrast to other effectors (chloramphenicol and aromatic solvents) for which exhibited a more reduced affinity. Structure-function studies of effectors indicate that the presence of aromatic rings as well as hydroxyl groups are determinants for TtgR binding. The binding of TtgR to its operator DNA does not alter the protein effector profile nor the effector binding stoichiometry. Moreover, we demonstrate here for the first time that the binding of a single effector molecule to the DNA-bound TtgR homodimer induces the dissociation of the repressor-operator complex. This provides important insight into the molecular mechanism of effector-mediated derepression.

  19. A DNA-templated silver nanocluster probe for label-free, turn-on fluorescence-based screening of homo-adenine binding molecules.

    PubMed

    Park, Ki Soo; Park, Hyun Gyu

    2015-02-15

    A novel, label-free, turn-on fluorescence strategy to detect molecules that bind to adenine-rich DNA sequences has been developed. The probe employs DNA-templated silver nanoclusters (DNA-AgNCs) as the key detection component. The new strategy relies on the formation of non-Watson-Crick homo-adenine DNA duplex, triggered by strong interactions with homo-adenine binding molecules, which brings a guanine-rich sequence in one strand close to DNA-AgNCs located on the opposite strand. This phenomenon transforms weakly fluorescent AgNCs into highly emissive species that display bright red fluorescence. Finally, we have shown that the new fluorescence turn-on strategy can be employed to detect coralyne, the most representative homo-adenine binding molecule that triggers formation of a non-Watson-Crick homo-adenine DNA duplex.

  20. Uncovering the peptide-binding specificities of HLA-C: a general strategy to determine the specificity of any MHC class I molecule.

    PubMed

    Rasmussen, Michael; Harndahl, Mikkel; Stryhn, Anette; Boucherma, Rachid; Nielsen, Lise Lotte; Lemonnier, François A; Nielsen, Morten; Buus, Søren

    2014-11-15

    MHC class I molecules (HLA-I in humans) present peptides derived from endogenous proteins to CTLs. Whereas the peptide-binding specificities of HLA-A and -B molecules have been studied extensively, little is known about HLA-C specificities. Combining a positional scanning combinatorial peptide library approach with a peptide-HLA-I dissociation assay, in this study we present a general strategy to determine the peptide-binding specificity of any MHC class I molecule. We applied this novel strategy to 17 of the most common HLA-C molecules, and for 16 of these we successfully generated matrices representing their peptide-binding motifs. The motifs prominently shared a conserved C-terminal primary anchor with hydrophobic amino acid residues, as well as one or more diverse primary and auxiliary anchors at P1, P2, P3, and/or P7. Matrices were used to generate a large panel of HLA-C-specific peptide-binding data and update our pan-specific NetMHCpan predictor, whose predictive performance was considerably improved with respect to peptide binding to HLA-C. The updated predictor was used to assess the specificities of HLA-C molecules, which were found to cover a more limited sequence space than HLA-A and -B molecules. Assessing the functional significance of these new tools, HLA-C*07:01 transgenic mice were immunized with stable HLA-C*07:01 binders; six of six tested stable peptide binders were immunogenic. Finally, we generated HLA-C tetramers and labeled human CD8(+) T cells and NK cells. These new resources should support future research on the biology of HLA-C molecules. The data are deposited at the Immune Epitope Database, and the updated NetMHCpan predictor is available at the Center for Biological Sequence Analysis and the Immune Epitope Database. Copyright © 2014 by The American Association of Immunologists, Inc.

  1. Dynamics of water around the complex structures formed between the KH domains of far upstream element binding protein and single-stranded DNA molecules.

    PubMed

    Chakraborty, Kaushik; Bandyopadhyay, Sanjoy

    2015-07-28

    Single-stranded DNA (ss-DNA) binding proteins specifically bind to the single-stranded regions of the DNA and protect it from premature annealing, thereby stabilizing the DNA structure. We have carried out atomistic molecular dynamics simulations of the aqueous solutions of two DNA binding K homology (KH) domains (KH3 and KH4) of the far upstream element binding protein complexed with two short ss-DNA segments. Attempts have been made to explore the influence of the formation of such complex structures on the microscopic dynamics and hydrogen bond properties of the interfacial water molecules. It is found that the water molecules involved in bridging the ss-DNA segments and the protein domains form a highly constrained thin layer with extremely retarded mobility. These water molecules play important roles in freezing the conformational oscillations of the ss-DNA oligomers and thereby forming rigid complex structures. Further, it is demonstrated that the effect of complexation on the slow long-time relaxations of hydrogen bonds at the interface is correlated with hindered motions of the surrounding water molecules. Importantly, it is observed that the highly restricted motions of the water molecules bridging the protein and the DNA components in the complexed forms originate from more frequent hydrogen bond reformations.

  2. Dynamics of water around the complex structures formed between the KH domains of far upstream element binding protein and single-stranded DNA molecules

    SciTech Connect

    Chakraborty, Kaushik; Bandyopadhyay, Sanjoy

    2015-07-28

    Single-stranded DNA (ss-DNA) binding proteins specifically bind to the single-stranded regions of the DNA and protect it from premature annealing, thereby stabilizing the DNA structure. We have carried out atomistic molecular dynamics simulations of the aqueous solutions of two DNA binding K homology (KH) domains (KH3 and KH4) of the far upstream element binding protein complexed with two short ss-DNA segments. Attempts have been made to explore the influence of the formation of such complex structures on the microscopic dynamics and hydrogen bond properties of the interfacial water molecules. It is found that the water molecules involved in bridging the ss-DNA segments and the protein domains form a highly constrained thin layer with extremely retarded mobility. These water molecules play important roles in freezing the conformational oscillations of the ss-DNA oligomers and thereby forming rigid complex structures. Further, it is demonstrated that the effect of complexation on the slow long-time relaxations of hydrogen bonds at the interface is correlated with hindered motions of the surrounding water molecules. Importantly, it is observed that the highly restricted motions of the water molecules bridging the protein and the DNA components in the complexed forms originate from more frequent hydrogen bond reformations.

  3. Dynamics of water around the complex structures formed between the KH domains of far upstream element binding protein and single-stranded DNA molecules

    NASA Astrophysics Data System (ADS)

    Chakraborty, Kaushik; Bandyopadhyay, Sanjoy

    2015-07-01

    Single-stranded DNA (ss-DNA) binding proteins specifically bind to the single-stranded regions of the DNA and protect it from premature annealing, thereby stabilizing the DNA structure. We have carried out atomistic molecular dynamics simulations of the aqueous solutions of two DNA binding K homology (KH) domains (KH3 and KH4) of the far upstream element binding protein complexed with two short ss-DNA segments. Attempts have been made to explore the influence of the formation of such complex structures on the microscopic dynamics and hydrogen bond properties of the interfacial water molecules. It is found that the water molecules involved in bridging the ss-DNA segments and the protein domains form a highly constrained thin layer with extremely retarded mobility. These water molecules play important roles in freezing the conformational oscillations of the ss-DNA oligomers and thereby forming rigid complex structures. Further, it is demonstrated that the effect of complexation on the slow long-time relaxations of hydrogen bonds at the interface is correlated with hindered motions of the surrounding water molecules. Importantly, it is observed that the highly restricted motions of the water molecules bridging the protein and the DNA components in the complexed forms originate from more frequent hydrogen bond reformations.

  4. Identification of Streptococcus mutans PAc peptide motif binding with human MHC class II molecules (DRB1*0802, *1101, *1401 and *1405).

    PubMed Central

    Senpuku, H; Yanagi, K; Nisizawa, T

    1998-01-01

    A surface protein antigen (PAc) of Streptococcus mutans, in particular the A-region of this PAc molecule, has been noted as a possible target in research for an effective dental caries vaccine. To identify the antigenic peptide binding to major histocompatibility complex (MHC) class II (HLA-DR) molecules in the A-region, we prepared a panel of overlapping synthetic peptides in the second unit of the A-region, and established that a simple enzyme-linked immunosorbent assay (ELISA) binding assay could be achieved by incubating the DR-crude. Binding to DR molecules of these peptides from nine donors was investigated by using the ELISA binding assay. It was revealed that the PAc(316-334) peptide bound more strongly to the HLA-DR molecule in seven out of nine subjects. In particular, DR8 (DRB1*0802), DR5 (DRB1*1101) and DR6 (DRB1*1402 and *1405), which bound strongly to PAc(316-334) peptide, were identified. Moreover, we synthesized glycine-substituted peptide analogues of the peptide and examined the binding motif of the binding region. As a result, the multiple binding motif in DR8, DR5 and DR6 was found in L-RV-K-A. It is suggested that a peptide vaccine for dental caries that is more effective for humans, with fewer adverse side-effects, could be designed by combining the multiple binding motif with the B-cell epitope to produce only the inhibiting antibody against dental caries. The peptide could therefore be useful for peptide vaccine development in the general human population. PMID:9824493

  5. Identification of Streptococcus mutans PAc peptide motif binding with human MHC class II molecules (DRB1*0802, *1101, *1401 and *1405).

    PubMed

    Senpuku, H; Yanagi, K; Nisizawa, T

    1998-11-01

    A surface protein antigen (PAc) of Streptococcus mutans, in particular the A-region of this PAc molecule, has been noted as a possible target in research for an effective dental caries vaccine. To identify the antigenic peptide binding to major histocompatibility complex (MHC) class II (HLA-DR) molecules in the A-region, we prepared a panel of overlapping synthetic peptides in the second unit of the A-region, and established that a simple enzyme-linked immunosorbent assay (ELISA) binding assay could be achieved by incubating the DR-crude. Binding to DR molecules of these peptides from nine donors was investigated by using the ELISA binding assay. It was revealed that the PAc(316-334) peptide bound more strongly to the HLA-DR molecule in seven out of nine subjects. In particular, DR8 (DRB1*0802), DR5 (DRB1*1101) and DR6 (DRB1*1402 and *1405), which bound strongly to PAc(316-334) peptide, were identified. Moreover, we synthesized glycine-substituted peptide analogues of the peptide and examined the binding motif of the binding region. As a result, the multiple binding motif in DR8, DR5 and DR6 was found in L-RV-K-A. It is suggested that a peptide vaccine for dental caries that is more effective for humans, with fewer adverse side-effects, could be designed by combining the multiple binding motif with the B-cell epitope to produce only the inhibiting antibody against dental caries. The peptide could therefore be useful for peptide vaccine development in the general human population.

  6. Evaluation of [(111/114m)In]CHX-A''-DTPA-ZHER2:342, an affibody ligand coniugate for targeting of HER2-expressing malignant tumors.

    PubMed

    Orlova, A; Rosik, D; Sandström, M; Lundqvist, H; Einarsson, L; Tolmachev, V

    2007-12-01

    Radionuclide imaging of the HER2 receptor, which is a target for trastuzumab therapy, can provide important diagnostic information. Further, targeting radionuclide therapy might be an option for treatment of HER2 expressing tumors. The phage-display selected Affibody ligand Z(HER2:342), which binds to HER2 with an affinity of 22 pM, may here play an important role. The small size of the Z(HER2:342), 7.5 kDa, enables quick tumor localization and fast blood clearance. Earlier, successful targeting of HER2-expressing xenografts using Z(HER2:342) labeled using [(111)In]benzyl-DTPA was reported. By changing to the CHX-A''-DTPA chelator, the stability and labeling kinetics of the radiometal-Z(HER2:342) conjugate can be improved. The aim of this study was to evaluate the labeling of the CHX-A''-DTPA-Z(HER2:342) conjugate with (111)In for diagnostic imaging and with (114m)In for locoregional radionuclide therapy. The isothiocyanate derivative of CHX-A''-DTPA was coupled to Z(HER2:342) in alkaline conditions at 37 degrees C. The conjugate was labeled with both (111)In and (114m)In and evaluated in vitro and in vivo. Labeling with (111)In and (114m)In provided >95% yield after 30 min at RT. Specific radioactivity was 0.5 and 12 MBq/nmol, for (114m)In and (111)In, respectively. The radiolabeled conjugates demonstrated specific binding to HER2 expressing SKOV-3 cells. In mice bearing SKOV-3 xenografts, the tumor uptake of [(111)In]CHX-A''-DTPA-Z(HER2:342) 4 h postinjection was 10.3+/-3.6% IA/g and tumor-to-blood ratio about 190. [(111)In]CHX-A''-DTPA-Z(HER2:342) is a promising candidate for the visualization of HER2 expression in malignant tumors. Labeled with (114m)In it could also be used for locoregional treatment of HER2 expressing tumors.

  7. A modern approach for epitope prediction: identification of foot-and-mouth disease virus peptides binding bovine leukocyte antigen (BoLA) class I molecules.

    PubMed

    Pandya, Mital; Rasmussen, Michael; Hansen, Andreas; Nielsen, Morten; Buus, Soren; Golde, William; Barlow, John

    2015-11-01

    Major histocompatibility complex (MHC) class Imolecules regulate adaptive immune responses through the presentation of antigenic peptides to CD8+ T cells. Polymorphisms in the peptide binding region of class I molecules determine peptide binding affinity and stability during antigen presentation, and different antigen peptide motifs are associated with specific genetic sequences of class I molecules. Understanding bovine leukocyte antigen (BoLA), peptide-MHC class I binding specificities may facilitate development of vaccines or reagents for quantifying the adaptive immune response to intracellular pathogens, such as foot-and-mouth disease virus (FMDV). Six synthetic BoLA class I (BoLA-I) molecules were produced, and the peptide binding motif was generated for five of the six molecules using a combined approach of positional scanning combinatorial peptide libraries (PSCPLs) and neural network-based predictions (NetMHCpan). The updated NetMHCpan server was used to predict BoLA-I binding peptides within the P1 structural polyprotein sequence of FMDV (strain A24 Cruzeiro) for Bo-LA-1*01901, BoLA-2*00801, BoLA-2*01201, and BoLA-4*02401. Peptide binding affinity and stability were determined for these BoLA-I molecules using the luminescent oxygen channeling immunoassay (LOCI) and scintillation proximity assay (SPA). The functional diversity of known BoLA alleles was predicted using theMHCcluster tool, and functional predictions for peptide motifs were compared to observed data from this and prior studies. The results of these analyses showed that BoLA alleles cluster into three distinct groups with the potential to define BBoLA supertypes.^ This streamlined approach identifies potential T cell epitopes from pathogens, such as FMDV, and provides insight into T cell immunity following infection or vaccination.

  8. New molecular motif for recognizing sialic acid using emissive lanthanide-macrocyclic polyazacarboxylate complexes: deprotonation of a coordinated water molecule controls specific binding.

    PubMed

    Ouchi, Kazuki; Saito, Shingo; Shibukawa, Masami

    2013-06-03

    A new molecular motif--lanthanide-macrocyclic polyazacarboxylate hexadentate complexes, Ln(3+)-ABNOTA--was found to specifically bind to sialic acid with strong emission enhancement and high affinity. The selectivity toward sialic acid over other monosaccharides was one of the highest among artificial receptors. Also, the novel binding mechanism was investigated in detail; binding selectivity is controlled by interactions between sialic acid and both the central metal and a hydroxyl group produced by deprotonation of a coordinated water molecule in the Ln(3+) complex.

  9. Application of differential scanning calorimetry to measure the differential binding of ions, water and protons in the unfolding of DNA molecules.

    PubMed

    Olsen, Chris M; Shikiya, Ronald; Ganugula, Rajkumar; Reiling-Steffensmeier, Calliste; Khutsishvili, Irine; Johnson, Sarah E; Marky, Luis A

    2016-05-01

    The overall stability of DNA molecules globally depends on base-pair stacking, base-pairing, polyelectrolyte effect and hydration contributions. In order to understand how they carry out their biological roles, it is essential to have a complete physical description of how the folding of nucleic acids takes place, including their ion and water binding. To investigate the role of ions, water and protons in the stability and melting behavior of DNA structures, we report here an experimental approach i.e., mainly differential scanning calorimetry (DSC), to determine linking numbers: the differential binding of ions (Δnion), water (ΔnW) and protons (ΔnH(+)) in the helix-coil transition of DNA molecules. We use DSC and temperature-dependent UV spectroscopic techniques to measure the differential binding of ions, water, and protons for the unfolding of a variety of DNA molecules: salmon testes DNA (ST-DNA), one dodecamer, one undecamer and one decamer duplexes, nine hairpin loops, and two triplexes. These methods can be applied to any conformational transition of a biomolecule. We determined complete thermodynamic profiles, including all three linking numbers, for the unfolding of each molecule. The favorable folding of a DNA helix results from a favorable enthalpy-unfavorable entropy compensation. DSC thermograms and UV melts as a function of salt, osmolyte and proton concentrations yielded releases of ions and water. Therefore, the favorable folding of each DNA molecule results from the formation of base-pair stacks and uptake of both counterions and water molecules. In addition, the triplex with C(+)GC base triplets yielded an uptake of protons. Furthermore, the folding of a DNA duplex is accompanied by a lower uptake of ions and a similar uptake of four water molecules as the DNA helix gets shorter. In addition, the oligomer duplexes and hairpin thermodynamic data suggest ion and water binding depends on the DNA sequence rather than DNA composition. Copyright

  10. Screening of paclitaxel-binding molecules from a library of random peptides displayed on T7 phage particles using paclitaxel-photoimmobilized resin.

    PubMed

    Aoki, Sota; Morohashi, Kengo; Sunoki, Takashi; Kuramochi, Kouji; Kobayashi, Susumu; Sugawara, Fumio

    2007-01-01

    Paclitaxel (Taxol), an effective anticancer agent, is known to bind to tubulin and induce tubulin polymerization. Several other binding proteins of paclitaxel, such as Bcl-2, heat shock proteins, and NSC-1, have also been reported. Here, we describe a T7 phage-based display to screen for paclitaxel-binding molecules from a random peptide library using paclitaxel-photoimmobilized TentaGel resin. Specific phage particles that bind the paclitaxel-immobilized resin were obtained. Among them, two phage clones included the same consensus amino acid sequence (KACGRTRVTS). Analysis of the protein database using BLAST revealed that a portion of this sequence is conserved in the zinc finger domain of human NFX1. Binding affinity of paclitaxel against the partial recombinant protein of NFX1 (424aa-876aa) was confirmed by pull-down assays and surface plasmon resonance analyses.

  11. Label-free optical detection of small-molecule compound microarrays immobilized on solid support using macromolecular scaffolds and subsequent protein binding reactions

    NASA Astrophysics Data System (ADS)

    Sun, Y. S.; Landry, J. P.; Fei, Y. Y.; Zhu, X. D.; Luo, J. T.; Wang, X. B.; Lam, K. S.

    2009-02-01

    Small-molecule microarrays composed of tens of thousands of distinct synthetic molecules, natural products, and their combinations/modifications provide a high-throughput platform for studying protein-ligand interactions. Immobilization of small molecule compounds on solid supports remains a challenge as widely varied small molecules generally lack unique chemical groups that readily react with singly or even multiply functionalized solid support. We explored two strategies for immobilizing small molecule compounds on epoxy-functionalized glass surface using primary-aminecontaining macromolecular scaffolds: bovine serum albumin (BSA) and amine-modified poly-vinyl alcohol (PVA). Small molecules with N-hydroxysuccinimide (NHS) groups were conjugated to BSA or amine-modified PVA. Small-molecule-BSA conjugates and small-molecule-PVA conjugates were subsequently immobilized on epoxy-functionalized glass slides through amine-epoxy reactions. Using an oblique-incidence reflectivity difference (OI-RD) scanning microscope as a label-free detector, we performed a comparative study of the effectiveness of BSA and PVA as macromolecular scaffolds for anchoring small molecule compounds in terms of conjugation efficiency, surface immobilization efficiency, effect of the scaffold on end-point and kinetics of subsequent binding reactions with protein probes.

  12. Interaction between Endothelial Protein C Receptor and Intercellular Adhesion Molecule 1 to Mediate Binding of Plasmodium falciparum-Infected Erythrocytes to Endothelial Cells

    PubMed Central

    Avril, Marion; Bernabeu, Maria; Benjamin, Maxwell; Brazier, Andrew Jay

    2016-01-01

    ABSTRACT Intercellular adhesion molecule 1 (ICAM-1) and the endothelial protein C receptor (EPCR) are candidate receptors for the deadly complication cerebral malaria. However, it remains unclear if Plasmodium falciparum parasites with dual binding specificity are involved in cytoadhesion or different parasite subpopulations bind in brain microvessels. Here, we investigated this issue by studying different subtypes of ICAM-1-binding parasite lines. We show that two parasite lines expressing domain cassette 13 (DC13) of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family have dual binding specificity for EPCR and ICAM-1 and further mapped ICAM-1 binding to the first DBLβ domain following the PfEMP1 head structure in both proteins. As PfEMP1 head structures have diverged between group A (EPCR binders) and groups B and C (CD36 binders), we also investigated how ICAM-1-binding parasites with different coreceptor binding traits influence P. falciparum-infected erythrocyte binding to endothelial cells. Whereas levels of binding to tumor necrosis factor alpha (TNF-α)-stimulated endothelial cells from the lung and brain by all ICAM-1-binding parasite lines increased, group A (EPCR and ICAM-1) was less dependent than group B (CD36 and ICAM-1) on ICAM-1 upregulation. Furthermore, both group A DC13 parasite lines had higher binding levels to brain endothelial cells (a microvascular niche with limited CD36 expression). This study shows that ICAM-1 is a coreceptor for a subset of EPCR-binding parasites and provides the first evidence of how EPCR and ICAM-1 interact to mediate parasite binding to both resting and TNF-α-activated primary brain and lung endothelial cells. PMID:27406562

  13. Combined quantum mechanics/molecular mechanics (QM/MM) simulations for protein-ligand complexes: free energies of binding of water molecules in influenza neuraminidase.

    PubMed

    Woods, Christopher J; Shaw, Katherine E; Mulholland, Adrian J

    2015-01-22

    The applicability of combined quantum mechanics/molecular mechanics (QM/MM) methods for the calculation of absolute binding free energies of conserved water molecules in protein/ligand complexes is demonstrated. Here, we apply QM/MM Monte Carlo simulations to investigate binding of water molecules to influenza neuraminidase. We investigate five different complexes, including those with the drugs oseltamivir and peramivir. We investigate water molecules in two different environments, one more hydrophobic and one hydrophilic. We calculate the free-energy change for perturbation of a QM to MM representation of the bound water molecule. The calculations are performed at the BLYP/aVDZ (QM) and TIP4P (MM) levels of theory, which we have previously demonstrated to be consistent with one another for QM/MM modeling. The results show that the QM to MM perturbation is significant in both environments (greater than 1 kcal mol(-1)) and larger in the more hydrophilic site. Comparison with the same perturbation in bulk water shows that this makes a contribution to binding. The results quantify how electronic polarization differences in different environments affect binding affinity and also demonstrate that extensive, converged QM/MM free-energy simulations, with good levels of QM theory, are now practical for protein/ligand complexes.

  14. Theoretical binding affinities and spectroscopy of complexes formed by cyclobis(paraquat- p-anthrancene) with some pharmaceutical molecules

    NASA Astrophysics Data System (ADS)

    Ren, Xin; Luo, Zhouyang; Du, Jinpei; Wu, Shi

    2010-05-01

    Theoretical investigation on the stabilities and spectroscopic properties of the complexes formed by cyciobis(paraquat- p-anthracene) with pharmaceutical molecules were performed using the semi-empirical PM3 and B3LYP/3-21G methods. Based on the B3LYP/3-21G optimized geometries, the energies of the complexes were calculated at B3LYP/6-31G( d) level. The binding energies of the complexes were computed after the correction of basis set superposition error (BSSE). The energy gaps of the complexes are decreased due to the formation of the hydrogen bonds. The stretching vibrations of the C-H bonds adjacent to the hydrogen bonds in the IR spectra of the complexes calculated with PM3 method are red-shifted compared with those of the host. The chemical shifts of α-C and β-C atoms in the complexes calculated at B3LYP/3-21G level are shifted downfield due to the formation of the hydrogen bonds and the electron-withdrawing effect of the nitrogen atoms. The aromaticities of the complexes are improved because of the enlargement of the conjugation system and the overlap of electron cloud based on the nuclear independent chemical shifts (NICS) calculated at B3LYP/3-21G level.

  15. High-Throughput Screening based Identification of Small Molecule Antagonists of Integrin CD11b/CD18 Ligand Binding

    PubMed Central

    Faridi, Mohd Hafeez; Maiguel, Dony; Brown, Brock T.; Suyama, Eigo; Barth, Constantinos J.; Hedrick, Michael; Vasile, Stefan; Sergienko, Eduard; Schürer, Stephan; Gupta, Vineet

    2010-01-01

    Binding of leukocyte specific integrin CD11b/CD18 to its physiologic ligands is important for the development of normal immune response in vivo. Integrin CD11b/CD18 is also a key cellular effector of various inflammatory and autoimmune diseases. However, small molecules selectively inhibiting the function of integrin CD11b/CD18 are currently lacking. We used a newly described cell-based high throughput screening assay to identify a number of highly potent antagonists of integrin CD11b/CD18 from chemical libraries containing >100,000 unique compounds. Computational analyses suggest that the identified compounds cluster into several different chemical classes. A number of the newly identified compounds blocked adhesion of wild-type mouse neutrophils to CD11b/CD18 ligand fibrinogen. Mapping the most active compounds against chemical fingerprints of known antagonists of related integrin CD11a/CD18 shows little structural similarity, suggesting that the newly identified compounds are novel and unique. PMID:20188705

  16. A small molecule inhibitor of tropomyosin dissociates actin binding from tropomyosin-directed regulation of actin dynamics

    PubMed Central

    Bonello, Teresa T.; Janco, Miro; Hook, Jeff; Byun, Alex; Appaduray, Mark; Dedova, Irina; Hitchcock-DeGregori, Sarah; Hardeman, Edna C.; Stehn, Justine R.; Böcking, Till; Gunning, Peter W.

    2016-01-01

    The tropomyosin family of proteins form end-to-end polymers along the actin filament. Tumour cells rely on specific tropomyosin-containing actin filament populations for growth and survival. To dissect out the role of tropomyosin in actin filament regulation we use the small molecule TR100 directed against the C terminus of the tropomyosin isoform Tpm3.1. TR100 nullifies the effect of Tpm3.1 on actin depolymerisation but surprisingly Tpm3.1 retains the capacity to bind F-actin in a cooperative manner. In vivo analysis also confirms that, in the presence of TR100, fluorescently tagged Tpm3.1 recovers normally into stress fibers. Assembling end-to-end along the actin filament is thereby not sufficient for tropomyosin to fulfil its function. Rather, regulation of F-actin stability by tropomyosin requires fidelity of information communicated at the barbed end of the actin filament. This distinction has significant implications for perturbing tropomyosin-dependent actin filament function in the context of anti-cancer drug development. PMID:26804624

  17. SCH529074, a small molecule activator of mutant p53, which binds p53 DNA binding domain (DBD), restores growth-suppressive function to mutant p53 and interrupts HDM2-mediated ubiquitination of wild type p53.

    PubMed

    Demma, Mark; Maxwell, Eugene; Ramos, Robert; Liang, Lianzhu; Li, Cheng; Hesk, David; Rossman, Randall; Mallams, Alan; Doll, Ronald; Liu, Ming; Seidel-Dugan, Cynthia; Bishop, W Robert; Dasmahapatra, Bimalendu

    2010-04-02

    Abrogation of p53 function occurs in almost all human cancers, with more than 50% of cancers harboring inactivating mutations in p53 itself. Mutation of p53 is indicative of highly aggressive cancers and poor prognosis. The vast majority of mutations in p53 occur in its core DNA binding domain (DBD) and result in inactivation of p53 by reducing its thermodynamic stability at physiological temperature. Here, we report a small molecule, SCH529074, that binds specifically to the p53 DBD in a saturable manner with an affinity of 1-2 microm. Binding restores wild type function to many oncogenic mutant forms of p53. This small molecule reactivates mutant p53 by acting as a chaperone, in a manner similar to that previously reported for the peptide CDB3. Binding of SCH529074 to the p53 DBD is specifically displaced by an oligonucleotide with a sequence derived from the p53-response element. In addition to reactivating mutant p53, SCH529074 binding inhibits ubiquitination of p53 by HDM2. We have also developed a novel variant of p53 by changing a single amino acid in the core domain of p53 (N268R), which abolishes binding of SCH529074. This amino acid change also inhibits HDM2-mediated ubiquitination of p53. Our novel findings indicate that through its interaction with p53 DBD, SCH529074 restores DNA binding activity to mutant p53 and inhibits HDM2-mediated ubiquitination.

  18. Amino Acid Sequences Mediating Vascular Cell Adhesion Molecule 1 Binding to Integrin Alpha 4: Homologous DSP Sequence Found for JC Polyoma VP1 Coat Protein

    PubMed Central

    Meyer, Michael Andrew

    2013-01-01

    The JC polyoma viral coat protein VP1 was analyzed for amino acid sequences homologies to the IDSP sequence which mediates binding of VLA-4 (integrin alpha 4) to vascular cell adhesion molecule 1. Although the full sequence was not found, a DSP sequence was located near the critical arginine residue linked to infectivity of the virus and binding to sialic acid containing molecules such as integrins (3). For the JC polyoma virus, a DSP sequence was found at residues 70, 71 and 72 with homology also noted for the mouse polyoma virus and SV40 virus. Three dimensional modeling of the VP1 molecule suggests that the DSP loop has an accessible site for interaction from the external side of the assembled viral capsid pentamer. PMID:24147211

  19. Amino Acid Sequences Mediating Vascular Cell Adhesion Molecule 1 Binding to Integrin Alpha 4: Homologous DSP Sequence Found for JC Polyoma VP1 Coat Protein.

    PubMed

    Meyer, Michael Andrew

    2013-01-01

    The JC polyoma viral coat protein VP1 was analyzed for amino acid sequences homologies to the IDSP sequence which mediates binding of VLA-4 (integrin alpha 4) to vascular cell adhesion molecule 1. Although the full sequence was not found, a DSP sequence was located near the critical arginine residue linked to infectivity of the virus and binding to sialic acid containing molecules such as integrins (3). For the JC polyoma virus, a DSP sequence was found at residues 70, 71 and 72 with homology also noted for the mouse polyoma virus and SV40 virus. Three dimensional modeling of the VP1 molecule suggests that the DSP loop has an accessible site for interaction from the external side of the assembled viral capsid pentamer.

  20. Targeting, bio distributive and tumor growth inhibiting characterization of anti-HER2 affibody coupling to liposomal doxorubicin using BALB/c mice bearing TUBO tumors.

    PubMed

    Akhtari, Javad; Rezayat, Seyed Mahdi; Teymouri, Manouchehr; Alavizadeh, Seyedeh Hoda; Gheybi, Fatemeh; Badiee, Ali; Jaafari, Mahmoud Reza

    2016-05-30

    Human epidermal growth factor receptor 2 (HER2) is overexpressed in 20-30% of breast cancer tumors. In the current investigation, we exploited such a feature and utilized an anti-HER2 affibody (ZHER2:477) in combination with a pegylated liposomal doxorubicin (PLD) for concurrent passive and active targeting of HER2 overexpressing TUBO tumor, using BALB/c mice. It was determined that the affibody coupled liposomes (affisomes) was capable of increasing doxorubicin (Dox) delivery to HER2+ cells (SK-BR-3 and TUBO cells), while transferring drug similarly as low as naïve PLD to HER2- MDA-MB-231 cells. This also resulted in selectively enhance cytotoxicity. The veracity of targeting was further assessed utilizing DiD lipophilic tracer model liposomes via competition assay. An approximated 10 ligand/liposome integration caused Dox delivery at 50% of maximal delivery capacity (Kd). Such integration did not alter Dox release in vitro, while it affected the serum clearance profile. Affibody integration to PLD increased drug concentration in tumor and led to significantly further augmentation of drug in liver and spleen compared to those of PLD. Overall, such differences led to prolonging the mice life spans as compared to PLD. Copyright © 2016. Published by Elsevier B.V.

  1. Crystal Structure of Mycobacterium tuberculosis H37Rv AldR (Rv2779c), a Regulator of the ald Gene: DNA BINDING AND IDENTIFICATION OF SMALL MOLECULE INHIBITORS.

    PubMed

    Dey, Abhishek; Shree, Sonal; Pandey, Sarvesh Kumar; Tripathi, Rama Pati; Ramachandran, Ravishankar

    2016-06-03

    Here we report the crystal structure of M. tuberculosis AldR (Rv2779c) showing that the N-terminal DNA-binding domains are swapped, forming a dimer, and four dimers are assembled into an octamer through crystal symmetry. The C-terminal domain is involved in oligomeric interactions that stabilize the oligomer, and it contains the effector-binding sites. The latter sites are 30-60% larger compared with homologs like MtbFFRP (Rv3291c) and can consequently accommodate larger molecules. MtbAldR binds to the region upstream to the ald gene that is highly up-regulated in nutrient-starved tuberculosis models and codes for l-alanine dehydrogenase (MtbAld; Rv2780). Further, the MtbAldR-DNA complex is inhibited upon binding of Ala, Tyr, Trp and Asp to the protein. Studies involving a ligand-binding site G131T mutant show that the mutant forms a DNA complex that cannot be inhibited by adding the amino acids. Comparative studies suggest that binding of the amino acids changes the relative spatial disposition of the DNA-binding domains and thereby disrupt the protein-DNA complex. Finally, we identified small molecules, including a tetrahydroquinoline carbonitrile derivative (S010-0261), that inhibit the MtbAldR-DNA complex. The latter molecules represent the very first inhibitors of a feast/famine regulatory protein from any source and set the stage for exploring MtbAldR as a potential anti-tuberculosis target.

  2. Ionized calcium‐binding adaptor molecule 1 positive macrophages and HO‐1 up‐regulation in intestinal muscularis resident macrophages

    PubMed Central

    Huizinga, Jan D.; Larsen, Jytte O.; Kirkeby, Svend

    2017-01-01

    ABSTRACT Small intestinal muscularis externa macrophages have been associated with interstitial cells of Cajal. They have been proposed to play various roles in motility disorders and to take part in a microbiota‐driven regulation of gastrointestinal motility. Our objective was to understand the reaction of resident macrophages of the musculature to a pro‐inflammatory stimulator, lipopolysaccharide (LPS). Mice were injected with LPS or saline and sacrificed after 6 hr. Whole mounts were stained with antibodies toward CD169, ionized calcium‐binding adaptor molecule 1 (iba1) (microglial/macrophage marker) and heme oxygenase‐1 (HO‐1). Cell densities were measured using unbiased stereology. Results: iba1pos cells showed an overall higher density than CD169pos and HO‐1pos cells. Most HO‐1pos and iba1pos cells were positive for CD 169 in serosa and at Auerbach's plexus (AP). At the deep muscular plexus, mainly iba1pos cells were present, and were mostly CD169neg; a few HO‐1pos cells were present. Conclusions: A new subset of resident macrophages in the intestinal muscularis externa was discovered, identified as iba1pos CD169neg. HO‐1 is constitutively present in most macrophages in serosa and at AP, suggesting a M2 phenotype. LPS‐treatment results in an up‐regulation of HO‐1pos/CD169neg cells in serosa and at AP. Anat Rec, 300:1114–1122, 2017. © 2016 The Authors. The Anatomical Record published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists PMID:27860408

  3. Ionized calcium-binding adaptor molecule 1 positive macrophages and HO-1 up-regulation in intestinal muscularis resident macrophages.

    PubMed

    Mikkelsen, Hanne B; Huizinga, Jan D; Larsen, Jytte O; Kirkeby, Svend

    2017-06-01

    Small intestinal muscularis externa macrophages have been associated with interstitial cells of Cajal. They have been proposed to play various roles in motility disorders and to take part in a microbiota-driven regulation of gastrointestinal motility. Our objective was to understand the reaction of resident macrophages of the musculature to a pro-inflammatory stimulator, lipopolysaccharide (LPS). Mice were injected with LPS or saline and sacrificed after 6 hr. Whole mounts were stained with antibodies toward CD169, ionized calcium-binding adaptor molecule 1 (iba1) (microglial/macrophage marker) and heme oxygenase-1 (HO-1). Cell densities were measured using unbiased stereology. iba1(pos) cells showed an overall higher density than CD169(pos) and HO-1(pos) cells. Most HO-1(pos) and iba1(pos) cells were positive for CD 169 in serosa and at Auerbach's plexus (AP). At the deep muscular plexus, mainly iba1(pos) cells were present, and were mostly CD169(neg) ; a few HO-1(pos) cells were present. A new subset of resident macrophages in the intestinal muscularis externa was discovered, identified as iba1(pos) CD169(neg) . HO-1 is constitutively present in most macrophages in serosa and at AP, suggesting a M2 phenotype. LPS-treatment results in an up-regulation of HO-1(pos) /CD169(neg) cells in serosa and at AP. Anat Rec, 300:1114-1122, 2017. © 2016 The Authors. The Anatomical Record published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists. © 2016 The Authors. The Anatomical Record published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists.

  4. The optimal binding sites of CH 4 and CO 2 molecules on the metal-organic framework MOF-5: ONIOM calculations

    NASA Astrophysics Data System (ADS)

    Pianwanit, Atchara; Kritayakornupong, Chinapong; Vongachariya, Arthit; Selphusit, Nattaya; Ploymeerusmee, Tanawut; Remsungnen, Tawun; Nuntasri, Duangamol; Fritzsche, Siegfried; Hannongbua, Supot

    2008-06-01

    Optimal binding sites and its corresponding binding energies between MOF-5 clusters and small guest molecules, CH 4 and CO 2, were investigated using the ONIOM method with different levels of quantum chemical calculations. The clusters were validated using three different sizes of the MOF-5 clusters, SINGLE, DOUBLE and TRIPLE consisting of (Zn 4O) 2(COOCH 3) 10(COO) 2C 6H 4, (Zn 4O) 3(COOCH 3) 14(COO) 4(C 6H 4) 2 and (Zn 4O) 4(COOCH 3) 18(COO) 6(C 6H 4) 3 units, respectively. Guest molecules were assigned to lie in the configurations parallel ( ∥) and perpendicular ( ⊥) to linker (LINK) and corner (CORN) domains of the clusters. The ONIOM(MP2/6-31G ∗∗:HF/6-31G ∗∗) with the corrections due to the basis set superposition errors was found to be the optimal choice for the investigation of these systems. Strong effects of cluster size were found for the CO 2/MOF-5 complexes, i.e., the SINGLE cluster is sufficient to represent interactions with CH 4, but the interaction with CO 2 requires the TRIPLE model. The optimal binding sites of guest molecules as well as their orientations in the cavity of the MOF-5 are CORN ⊥ for both CH 4 and CO 2 with the corresponding binding energies of -3.64 and -9.27 kJ/mol, respectively.

  5. Structure-activity studies on the fluorescent indicator in a displacement assay for the screening of small molecules binding to RNA.

    PubMed

    Umemoto, Shiori; Im, Seongwang; Zhang, Jinhua; Hagihara, Masaki; Murata, Asako; Harada, Yasue; Fukuzumi, Takeo; Wazaki, Takahiro; Sasaoka, Shin-ichi; Nakatani, Kazuhiko

    2012-08-06

    A series of xanthone and thioxanthone derivatives with aminoalkoxy substituents were synthesized as fluorescent indicators for a displacement assay in the study of small-molecule-RNA interactions. The RNA-binding properties of these molecules were investigated in terms of the improved binding selectivity to the loop region in the RNA secondary structure relative to 2,7-bis(2-aminoethoxy)xanthone (X2S) by fluorimetric titration and displacement assay. An 11-mer double-stranded RNA and a hairpin RNA mimicking the stem loop IIB of Rev response element (RRE) RNA of HIV-1 mRNA were used. The X2S derivatives with longer aminoalkyl substituents showed a higher affinity to the double-stranded RNA than the parent molecule. Introduction of a methyl group on the aminoethoxy moiety of X2S effectively modulated the selectivity to the RNA secondary structure. Methyl group substitution at the C1' position suppressed the binding to the loop regions. Substitution with two methyl groups on the amino nitrogen atom resulted in reducing the affinity to the double-stranded region by a factor of 40%. The effect of methyl substitution on the amino nitrogen atom was also observed for a thioxanthone derivative. Titration experiments, however, suggested that thioxanthone derivatives showed a more prominent tendency of multiple binding to RNA than xanthone derivatives. The selectivity index calculated from the affinity to the double-stranded and loop regions suggested that the N,N-dimethyl derivative of X2S would be suitable for the screening of small molecules binding to RRE. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. A study of the molecular mechanism of binding kinetics and long residence times of human CCR5 receptor small molecule allosteric ligands

    PubMed Central

    Swinney, David C; Beavis, Paul; Chuang, Kai-Ting; Zheng, Yue; Lee, Ina; Gee, Peter; Deval, Jerome; Rotstein, David M; Dioszegi, Marianna; Ravendran, Palani; Zhang, Jun; Sankuratri, Surya; Kondru, Rama; Vauquelin, Georges

    2014-01-01

    BACKGROUND AND PURPOSE The human CCR5 receptor is a co-receptor for HIV-1 infection and a target for anti-viral therapy. A greater understanding of the binding kinetics of small molecule allosteric ligand interactions with CCR5 will lead to a better understanding of the binding process and may help discover new molecules that avoid resistance. EXPERIMENTAL APPROACH Using [3H] maraviroc as a radioligand, a number of different binding protocols were employed in conjunction with simulations to determine rate constants, kinetic mechanism and mutant kinetic fingerprints for wild-type and mutant human CCR5 with maraviroc, aplaviroc and vicriviroc. KEY RESULTS Kinetic characterization of maraviroc binding to the wild-type CCR5 was consistent with a two-step kinetic mechanism that involved an initial receptor–ligand complex (RA), which transitioned to a more stable complex, R'A, with at least a 13-fold increase in affinity. The dissociation rate from R'A, k−2, was 1.2 × 10−3 min−1. The maraviroc time-dependent transition was influenced by F85L, W86A, Y108A, I198A and Y251A mutations of CCR5. CONCLUSIONS AND IMPLICATIONS The interaction between maraviroc and CCR5 proceeded according to a multi-step kinetic mechanism, whereby initial mass action binding and later reorganizations of the initial maraviroc–receptor complex lead to a complex with longer residence time. Site-directed mutagenesis identified a kinetic fingerprint of residues that affected the binding kinetics, leading to the conclusion that allosteric ligand binding to CCR5 involved the rearrangement of the binding site in a manner specific to each allosteric ligand. PMID:24628038

  7. A strategy for the incorporation of water molecules present in a ligand binding site into a three-dimensional quantitative structure--activity relationship analysis.

    PubMed

    Pastor, M; Cruciani, G; Watson, K A

    1997-12-05

    Water present in a ligand binding site of a protein has been recognized to play a major role in ligand-protein interactions. To date, rational drug design techniques do not usually incorporate the effect of these water molecules into the design strategy. This work represents a new strategy for including water molecules into a three-dimensional quantitative structure-activity relationship analysis using a set of glucose analogue inhibitors of glycogen phosphorylase (GP). In this series, the structures of the ligand-enzyme complexes have been solved by X-ray crystallography, and the positions of the ligands and the water molecules at the ligand binding site are known. For the structure-activity analysis, some water molecules adjacent to the ligands were included into an assembly which encompasses both the inhibitor and the water involved in the ligand-enzyme interaction. The mobility of some water molecules at the ligand binding site of GP gives rise to differences in the ligand-water assembly which have been accounted for using a simulation study involving force-field energy calculations. The assembly of ligand plus water was used in a GRID/GOLPE analysis, and the models obtained compare favorably with equivalent models when water was excluded. Both models were analyzed in detail and compared with the crystallographic structures of the ligand-enzyme complexes in order to evaluate their ability to reproduce the experimental observations. The results demonstrate that incorporation of water molecules into the analysis improves the predictive ability of the models and makes them easier to interpret. The information obtained from interpretation of the models is in good agreement with the conclusions derived from the structural analysis of the complexes and offers valuable insights into new characteristics of the ligands which may be exploited for the design of more potent inhibitors.

  8. Role of Conserved Disulfide Bridges and Aromatic Residues in Extracellular Loop 2 of Chemokine Receptor CCR8 for Chemokine and Small Molecule Binding.

    PubMed

    Barington, Line; Rummel, Pia C; Lückmann, Michael; Pihl, Heidi; Larsen, Olav; Daugvilaite, Viktorija; Johnsen, Anders H; Frimurer, Thomas M; Karlshøj, Stefanie; Rosenkilde, Mette M

    2016-07-29

    Chemokine receptors play important roles in the immune system and are linked to several human diseases. The initial contact of chemokines with their receptors depends on highly specified extracellular receptor features. Here we investigate the importance of conserved extracellular disulfide bridges and aromatic residues in extracellular loop 2 (ECL-2) for ligand binding and activation in the chemokine receptor CCR8. We used inositol 1,4,5-trisphosphate accumulation and radioligand binding experiments to determine the impact of receptor mutagenesis on both chemokine and small molecule agonist and antagonist binding and action in CCR8. We find that the seven-transmembrane (TM) receptor conserved disulfide bridge (7TM bridge) linking transmembrane helix III (TMIII) and ECL-2 is crucial for chemokine and small molecule action, whereas the chemokine receptor conserved disulfide bridge between the N terminus and TMVII is needed only for chemokines. Furthermore, we find that two distinct aromatic residues in ECL-2, Tyr(184) (Cys + 1) and Tyr(187) (Cys + 4), are crucial for binding of the CC chemokines CCL1 (agonist) and MC148 (antagonist), respectively, but not for small molecule binding. Finally, using in silico modeling, we predict an aromatic cluster of interaction partners for Tyr(187) in TMIV (Phe(171)) and TMV (Trp(194)). We show in vitro that these residues are crucial for the binding and action of MC148, thus supporting their participation in an aromatic cluster with Tyr(187) This aromatic cluster appears to be present in a large number of CC chemokine receptors and thereby could play a more general role to be exploited in future drug development targeting these receptors. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. Role of Conserved Disulfide Bridges and Aromatic Residues in Extracellular Loop 2 of Chemokine Receptor CCR8 for Chemokine and Small Molecule Binding*

    PubMed Central

    Barington, Line; Rummel, Pia C.; Lückmann, Michael; Pihl, Heidi; Larsen, Olav; Daugvilaite, Viktorija; Johnsen, Anders H.; Frimurer, Thomas M.; Karlshøj, Stefanie; Rosenkilde, Mette M.

    2016-01-01

    Chemokine receptors play important roles in the immune system and are linked to several human diseases. The initial contact of chemokines with their receptors depends on highly specified extracellular receptor features. Here we investigate the importance of conserved extracellular disulfide bridges and aromatic residues in extracellular loop 2 (ECL-2) for ligand binding and activation in the chemokine receptor CCR8. We used inositol 1,4,5-trisphosphate accumulation and radioligand binding experiments to determine the impact of receptor mutagenesis on both chemokine and small molecule agonist and antagonist binding and action in CCR8. We find that the seven-transmembrane (TM) receptor conserved disulfide bridge (7TM bridge) linking transmembrane helix III (TMIII) and ECL-2 is crucial for chemokine and small molecule action, whereas the chemokine receptor conserved disulfide bridge between the N terminus and TMVII is needed only for chemokines. Furthermore, we find that two distinct aromatic residues in ECL-2, Tyr184 (Cys + 1) and Tyr187 (Cys + 4), are crucial for binding of the CC chemokines CCL1 (agonist) and MC148 (antagonist), respectively, but not for small molecule binding. Finally, using in silico modeling, we predict an aromatic cluster of interaction partners for Tyr187 in TMIV (Phe171) and TMV (Trp194). We show in vitro that these residues are crucial for the binding and action of MC148, thus supporting their participation in an aromatic cluster with Tyr187. This aromatic cluster appears to be present in a large number of CC chemokine receptors and thereby could play a more general role to be exploited in future drug development targeting these receptors. PMID:27226537

  10. Two MHC class I molecules associated with elite control of immunodeficiency virus replication, Mamu-B*08 and HLA-B*2705, bind peptides with sequence similarity1

    PubMed Central

    Loffredo, John T.; Sidney, John; Bean, Alex T.; Beal, Dominic R.; Bardet, Wilfried; Wahl, Angela; Hawkins, Oriana E.; Piaskowski, Shari; Wilson, Nancy A.; Hildebrand, William H.; Watkins, David I.; Sette, Alessandro

    2009-01-01

    HLA-B27- and -B57-positive HIV-infected humans have long been associated with control of HIV replication, implying that CD8+ T cell responses contribute to control of viral replication. In a similar fashion, fifty percent of Mamu-B*08-positive Indian rhesus macaques control SIVmac239 replication and become elite controllers with chronic phase viremia below 1,000 vRNA copies/ml. Interestingly, Mamu-B*08-restricted SIV-derived epitopes appeared to match the peptide binding profile for HLA-B*2705 in humans. We, therefore, defined a detailed peptide-binding motif for Mamu-B*08 and investigated binding similarities between the macaque and human MHC class I molecules. Analysis of a panel of almost 900 peptides revealed that despite substantial sequence differences between Mamu-B*08 and HLA-B*2705, the peptide-binding repertoires of these two MHC class I molecules share a remarkable degree of overlap. Detailed knowledge of the Mamu-B*08 peptide-binding motif enabled us to identify six additional novel Mamu-B*08-restricted SIV-specific CD8+ T cell immune responses directed against epitopes in Gag, Vpr, and Env. All 13 Mamu-B*08-restricted epitopes contain an R at the position 2 primary anchor, and 10 also possess either R or K at the N-terminus. Such dibasic peptides are less prone to cellular degradation. This work highlights the relevance of the Mamu-B*08-positive SIV-infected Indian rhesus macaque as a model to examine elite control of immunodeficiency virus replication. The remarkable similarity of the peptide-binding motifs and repertoires for Mamu-B*08 and HLA-B*2705 suggests that the nature of the peptide bound by the MHC class I molecule may play an important role in control of immunodeficiency virus replication. PMID:19494300

  11. Time-Resolved Fluorescence Resonance Energy Transfer Assay for Discovery of Small-Molecule Inhibitors of Methyl-CpG Binding Domain Protein 2.

    PubMed

    Wyhs, Nicolas; Walker, David; Giovinazzo, Hugh; Yegnasubramanian, Srinivasan; Nelson, William G

    2014-08-01

    Methylated DNA binding proteins such as Methyl-CpG Binding Domain Protein 2 (MBD2) can transduce DNA methylation alterations into a repressive signal by recruiting transcriptional co-repressor complexes. Interfering with MBD2 could lead to reactivation of tumor suppressor genes and therefore represents an attractive strategy for epigenetic therapy. We developed and compared fluorescence polarization (FP) and time-resolved fluorescence resonance energy transfer (TR-FRET)-based high-throughput screening (HTS) assays to identify small-molecule inhibitors of the interaction between the methyl binding domain of MBD2 (MBD2-MBD) and methylated DNA. Although both assays performed well in 96-well format, the TR-FRET assay (Z' factor = 0.58) emerged as a superior screening strategy compared with FP (Z' factor = 0.08) when evaluated in an HTS 384-well plate format. Using TR-FRET, we screened the Sigma LOPAC library for MBD2-MBD inhibitors and identified four compounds that also validated in a dose-response series. This included two known DNA intercalators (mitoxantrone and idarubicin) among two other inhibitory compounds (NF449 and aurintricarboxylic acid). All four compounds also inhibited the binding of SP-1, a transcription factor with a GC-rich binding sequence, to a methylated oligonucleotide, demonstrating that the activity was nonspecific. Our results provide proof of principle for using TR-FRET-based HTS to identify small-molecule inhibitors of MBD2 and other DNA-protein interactions. © 2014 Society for Laboratory Automation and Screening.

  12. Definition of an extended MHC class II-peptide binding motif for the autoimmune disease-associated Lewis rat RT1.BL molecule.

    PubMed

    Wauben, M H; van der Kraan, M; Grosfeld-Stulemeyer, M C; Joosten, I

    1997-02-01

    The Lewis rat, an inbred rat strain susceptible to several well-characterized experimental autoimmune diseases, provides a good model to study peptide-mediated immunotherapy. Peptide immunotherapy focussing on the modulation of T cell responses by interfering with TCR-peptide-MHC complex formation requires the elucidation of the molecular basis of TCR-peptide-MHC interactions for an efficient design of modulatory peptides. In the Lewis rat most autoimmune-associated CD4+ T cell responses are MHC class II RT1.BL restricted. In this study, the characteristics of RT1.BL-peptide interactions were explored. A series of substitution analogs of two Lewis rat T cell epitopes was examined in a direct peptide-MHC binding assay on isolated RT1.BL molecules. Furthermore, other autoimmune-related as well as non-disease-related T cell epitopes were tested in the binding assay. This has led to the definition of an extended RT1.BL-peptide binding motif. The RT1.BL-peptide binding motif established in this study is the first described rat MHC-peptide binding motif based on direct MHC-peptide binding experiments. To predict good or intermediate RT1.BL binding peptides, T cell epitope search profiles were deduced from this motif. The motif and search profiles will greatly facilitate the prediction of modulatory peptides based on autoimmune-associated T cell epitopes and the identification of target structures in experimental autoimmune diseases in Lewis rats.

  13. Role of curdlan sulfate in the binding of HIV-1 gp120 to CD4 molecules and the production of gp120-mediated TNF-alpha.

    PubMed

    Takeda-Hirokawa, N; Neoh, L P; Akimoto, H; Kaneko, H; Hishikawa, T; Sekigawa, I; Hashimoto, H; Hirose, S; Murakami, T; Yamamoto, N; Mimura, T; Kaneko, Y

    1997-01-01

    To clarify the mechanism by which curdlan sulfate (CRDS) inhibits human immunodeficiency virus (HIV)-1 infection, we examined its influence on the binding of gp120 to CD4 molecules on T cells and macrophages, as well as on the production of TNF-alpha by gp120-stimulated macrophages (which promotes HIV-1 replication). CRDS treatment of cells not only inhibited the binding of HIV-1 gp120 to CD4+ cells, but also inhibited TNF-alpha production induced by gp120. Inhibition of HIV-1 infection by CRDS may be related to these two actions.

  14. Discovery of Small-Molecule Human Immunodeficiency Virus Type 1 Entry Inhibitors That Target the gp120-Binding Domain of CD4†

    PubMed Central

    Yang, Quan-en; Stephen, Andrew G.; Adelsberger, Joseph W.; Roberts, Paula E.; Zhu, Weimin; Currens, Michael J.; Feng, Yaxiong; Crise, Bruce J.; Gorelick, Robert J.; Rein, Alan R.; Fisher, Robert J.; Shoemaker, Robert H.; Sei, Shizuko

    2005-01-01

    The interaction between human immunodeficiency virus type 1 (HIV-1) gp120 and the CD4 receptor is highly specific and involves relatively small contact surfaces on both proteins according to crystal structure analysis. This molecularly conserved interaction presents an excellent opportunity for antiviral targeting. Here we report a group of pentavalent antimony-containing small molecule compounds, NSC 13778 (molecular weight, 319) and its analogs, which exert a potent anti-HIV activity. These compounds block the entry of X4-, R5-, and X4/R5-tropic HIV-1 strains into CD4+ cells but show little or no activity in CD4-negative cells or against vesicular stomatitis virus-G pseudotyped virions. The compounds compete with gp120 for binding to CD4: either immobilized on a solid phase (soluble CD4) or on the T-cell surface (native CD4 receptor) as determined by a competitive gp120 capture enzyme-linked immunosorbent assay or flow cytometry. NSC 13778 binds to an N-terminal two-domain CD4 protein, D1/D2 CD4, immobilized on a surface plasmon resonance sensor chip, and dose dependently reduces the emission intensity of intrinsic tryptophan fluorescence of D1/D2 CD4, which contains two of the three tryptophan residues in the gp120-binding domain. Furthermore, T cells incubated with the compounds alone show decreased reactivity to anti-CD4 monoclonal antibodies known to recognize the gp120-binding site. In contrast to gp120-binders that inhibit gp120-CD4 interaction by binding to gp120, these compounds appear to disrupt gp120-CD4 contact by targeting the specific gp120-binding domain of CD4. NSC 13778 may represent a prototype of a new class of HIV-1 entry inhibitors that can break into the gp120-CD4 interface and mask the gp120-binding site on the CD4 molecules, effectively repelling incoming virions. PMID:15857997

  15. Binding of an octylglucoside detergent molecule in the second substrate (S2) site of LeuT establishes an inhibitor-bound conformation.

    PubMed

    Quick, Matthias; Winther, Anne-Marie Lund; Shi, Lei; Nissen, Poul; Weinstein, Harel; Javitch, Jonathan A

    2009-04-07

    The first crystal structure of the neurotransmitter/sodium symporter homolog LeuT revealed an occluded binding pocket containing leucine and 2 Na(+); later structures showed tricyclic antidepressants (TCAs) in an extracellular vestibule approximately 11 A above the bound leucine and 2 Na(+). We recently found this region to be a second binding (S2) site and that binding of substrate to this site triggers Na(+)-coupled substrate symport. Here, we show a profound inhibitory effect of n-octyl-beta-d-glucopyranoside (OG), the detergent used for LeuT crystallization, on substrate binding to the S2 site. In parallel, we determined at 2.8 A the structure of LeuT-E290S, a mutant that, like LeuT-WT, binds 2 substrate molecules. This structure was similar to that of WT and clearly revealed an OG molecule in the S2 site. We also observed electron density at the S2 site in LeuT-WT crystals, and this also was accounted for by an OG molecule in that site. Computational analyses, based on the available crystal structures of LeuT, indicated the nature of structural arrangements in the extracellular region of LeuT that differentiate the actions of substrates from inhibitors bound in the S2 site. We conclude that the current LeuT crystal structures, all of which have been solved in OG, represent functionally blocked forms of the transporter, whereas a substrate bound in the S2 site will promote a different state that is essential for Na(+)-coupled symport.

  16. N-terminal aliphatic residues dictate the structure, stability, assembly, and small molecule binding of the coiled-coil region of cartilage oligomeric matrix protein.

    PubMed

    Gunasekar, Susheel K; Asnani, Mukta; Limbad, Chandani; Haghpanah, Jennifer S; Hom, Wendy; Barra, Hanna; Nanda, Soumya; Lu, Min; Montclare, Jin Kim

    2009-09-15

    The coiled-coil domain of cartilage oligomeric matrix protein (COMPcc) assembles into a homopentamer that naturally recognizes the small molecule 1,25-dihydroxyvitamin D(3) (vit D). To identify the residues critical for the structure, stability, oligomerization, and binding to vit D as well as two other small molecules, all-trans-retinol (ATR) and curcumin (CCM), here we perform an alanine scanning mutagenesis study. Ten residues lining the hydrophobic pocket of COMPcc were mutated into alanine; of the mutated residues, the N-terminal aliphatic residues L37, L44, V47, and L51 are responsible for maintaining the structure and function. Furthermore, two polar residues, T40 and Q54, within the N-terminal region when converted into alanine improve the alpha-helical structure, stability, and self-assembly behavior. Helical stability, oligomerization, and binding appear to be linked in a manner in which mutations that abolish helical structure and assembly bind poorly to vit D, ATR, and CCM. These results provide not only insight into COMPcc and its functional role but also useful guidelines for the design of stable, pentameric coiled-coils capable of selectively storing and delivering various small molecules.

  17. Amyloid-binding small molecules efficiently block SEVI (semen-derived enhancer of virus infection)- and semen-mediated enhancement of HIV-1 infection.

    PubMed

    Olsen, Joanna S; Brown, Caitlin; Capule, Christina C; Rubinshtein, Mark; Doran, Todd M; Srivastava, Rajesh K; Feng, Changyong; Nilsson, Bradley L; Yang, Jerry; Dewhurst, Stephen

    2010-11-12

    Semen was recently shown to contain amyloid fibrils formed from a self-assembling peptide fragment of the protein prostatic acid phosphatase. These amyloid fibrils, termed semen-derived enhancer of virus infection, or SEVI, have been shown to strongly enhance HIV infectivity and may play an important role in sexual transmission of HIV, making them a potential microbicide target. One novel approach to target these fibrils is the use of small molecules known to intercalate into the structure of amyloid fibrils, such as derivatives of thioflavin-T. Here, we show that the amyloid-binding small molecule BTA-EG(6) (the hexa(ethylene glycol) derivative of benzothiazole aniline) is able to bind SEVI fibrils and effectively inhibit both SEVI-mediated and semen-mediated enhancement of HIV infection. BTA-EG(6) also blocks the interactions of SEVI with HIV-1 virions and HIV-1 target cells but does not cause any inflammation or toxicity to cervical epithelial cells. These results suggest that an amyloid-binding small molecule may have utility as a microbicide, or microbicidal supplement, for HIV-1.

  18. Amyloid-binding Small Molecules Efficiently Block SEVI (Semen-derived Enhancer of Virus Infection)- and Semen-mediated Enhancement of HIV-1 Infection*

    PubMed Central

    Olsen, Joanna S.; Brown, Caitlin; Capule, Christina C.; Rubinshtein, Mark; Doran, Todd M.; Srivastava, Rajesh K.; Feng, Changyong; Nilsson, Bradley L.; Yang, Jerry; Dewhurst, Stephen

    2010-01-01

    Semen was recently shown to contain amyloid fibrils formed from a self-assembling peptide fragment of the protein prostatic acid phosphatase. These amyloid fibrils, termed semen-derived enhancer of virus infection, or SEVI, have been shown to strongly enhance HIV infectivity and may play an important role in sexual transmission of HIV, making them a potential microbicide target. One novel approach to target these fibrils is the use of small molecules known to intercalate into the structure of amyloid fibrils, such as derivatives of thioflavin-T. Here, we show that the amyloid-binding small molecule BTA-EG6 (the hexa(ethylene glycol) derivative of benzothiazole aniline) is able to bind SEVI fibrils and effectively inhibit both SEVI-mediated and semen-mediated enhancement of HIV infection. BTA-EG6 also blocks the interactions of SEVI with HIV-1 virions and HIV-1 target cells but does not cause any inflammation or toxicity to cervical epithelial cells. These results suggest that an amyloid-binding small molecule may have utility as a microbicide, or microbicidal supplement, for HIV-1. PMID:20833717

  19. Small molecule inhibitor of antigen binding and presentation by HLA-DR2b as a therapeutic strategy for the treatment of multiple sclerosis.

    PubMed

    Ji, Niannian; Somanaboeina, Animesh; Dixit, Aakanksha; Kawamura, Kazuyuki; Hayward, Neil J; Self, Christopher; Olson, Gary L; Forsthuber, Thomas G

    2013-11-15

    The strong association of HLA-DR2b (DRB1*1501) with multiple sclerosis (MS) suggests this molecule as prime target for specific immunotherapy. Inhibition of HLA-DR2b-restricted myelin-specific T cells has the potential to selectively prevent CNS pathology mediated by these MHC molecules without undesired global immunosuppression. In this study, we report development of a highly selective small molecule inhibitor of peptide binding and presentation by HLA-DR2b. PV-267, the candidate molecule used in these studies, inhibited cytokine production and proliferation of myelin-specific HLA-DR2b-restricted T cells. PV-267 had no significant effect on T cell responses mediated by other MHC class II molecules, including HLA-DR1, -DR4, or -DR9. Importantly, PV-267 did not induce nonspecific immune activation of human PBMC. Lastly, PV-267 showed treatment efficacy both in preventing experimental autoimmune encephalomyelitis and in treating established disease. The results suggest that blocking the MS-associated HLA-DR2b allele with small molecule inhibitors may be a promising therapeutic strategy for the treatment of MS.

  20. The binding of chondroitin sulfate to pleiotrophin/heparin-binding growth-associated molecule is regulated by chain length and oversulfated structures.

    PubMed

    Maeda, Nobuaki; Fukazawa, Nobuna; Hata, Toshihiro

    2006-02-24

    Pleiotrophin is an 18-kDa heparin-binding growth factor, which uses chondroitin sulfate (CS) proteoglycan, PTPzeta as a receptor. It has been suggested that the D-type structure (GlcA(2S)beta1-3GalNAc(6S)) in CS contributes to the high affinity binding between PTPzeta and pleiotrophin. Here, we analyzed the interaction of shark cartilage CS-D with pleiotrophin using a surface plasmon resonance biosensor to reveal the importance of D-type structure. CS-D was partially digested with chondroitinase ABC, and fractionated using a Superdex 75pg column. The > or =18-mer CS fractions showed significant binding to pleiotrophin, and the longer fractions had stronger affinity for pleiotrophin than the shorter ones. The approximately 46-mer CS fraction bound to densely immobilized pleiotrophin with high affinity (K(D) = approximately 30 nM), and the binding reactions fitted the bivalent analyte model. However, when the density of the immobilized pleiotrophin was lowered, the strength of affinity remarkably decreased (K(D) = approximately 2.5 microM), and the reactions no longer fitted the model and were considered to be monovalent binding. The 20 approximately 24-mer fractions showed low affinity binding to densely immobilized pleiotrophin (K(D) = 3 approximately 20 microM), which seemed to be monovalent. When approximately 22-mer CS oligosaccharides were fractionated by strong anion exchange HPLC, each fraction differed in affinity for pleiotrophin (K(D) = 0.36 approximately >10 microM), and the affinity correlated with the amounts of D- and E- (GlcAbeta1-3GalNAc(4S,6S)) type oversulfated structures. These results suggest that the binding of pleiotrophin to CS is regulated by multivalency with CS approximately 20 mer as a unit and by the amounts of oversulfated structures.

  1. Detection of 3D atomic similarities and their use in the discrimination of small molecule protein-binding sites.

    PubMed

    Najmanovich, Rafael; Kurbatova, Natalja; Thornton, Janet

    2008-08-15

    Current computational methods for the prediction of function from structure are restricted to the detection of similarities and subsequent transfer of functional annotation. In a significant minority of cases, global sequence or structural (fold) similarities do not provide clues about protein function. In these cases, one alternative is to detect local binding site similarities. These may still reflect more distant evolutionary relationships as well as unique physico-chemical constraints necessary for binding similar ligands, thus helping pinpoint the function. In the present work, we ask the following question: is it possible to discriminate within a dataset of non-homologous proteins those that bind similar ligands based on their binding site similarities? We implement a graph-matching-based method for the detection of 3D atomic similarities introducing some simplifications that allow us to extend its applicability to the analysis of large allatom binding site models. This method, called IsoCleft, does not require atoms to be connected either in sequence or space. We apply the method to a cognate-ligand bound dataset of non-homologous proteins. We define a family of binding site models with decreasing knowledge about the identity of the ligand-interacting atoms to uncouple the questions of predicting the location of the binding site and detecting binding site similarities. Furthermore, we calculate the individual contributions of binding site size, chemical composition and geometry to prediction performance. We find that it is possible to discriminate between different ligand-binding sites. In other words, there is a certain uniqueness in the set of atoms that are in contact to specific ligand scaffolds. This uniqueness is restricted to the atoms in close proximity of the ligand in which case, size and chemical composition alone are sufficient to discriminate binding sites. Discrimination ability decreases with decreasing knowledge about the identity of the

  2. Live-cell p53 single-molecule binding is modulated by C-terminal acetylation and correlates with transcriptional activity.

    PubMed

    Loffreda, Alessia; Jacchetti, Emanuela; Antunes, Sofia; Rainone, Paolo; Daniele, Tiziana; Morisaki, Tatsuya; Bianchi, Marco E; Tacchetti, Carlo; Mazza, Davide

    2017-08-22

    Live-cell microscopy has highlighted that transcription factors bind transiently to chromatin but it is not clear if the duration of these binding interactions can be modulated in response to an activation stimulus, and if such modulation can be controlled by post-translational modifications of the transcription factor. We address this question for the tumor suppressor p53 by combining live-cell single-molecule microscopy and single cell in situ measurements of transcription and we show that p53-binding kinetics are modulated following genotoxic stress. The modulation of p53 residence times on chromatin requires C-terminal acetylation-a classical mark for transcriptionally active p53-and correlates with the induction of transcription of target genes such as CDKN1a. We propose a model in which the modification state of the transcription factor determines the coupling between transcription factor abundance and transcriptional activity by tuning the transcription factor residence time on target sites.Both transcription binding kinetics and post-translational modifications of transcription factors are thought to play a role in the modulation of transcription. Here the authors use single-molecule tracking to directly demonstrate that p53 acetylation modulates promoter residence time and transcriptional activity.

  3. Discovery of a small-molecule HIV-1 integrase inhibitor-binding site | Center for Cancer Research

    Cancer.gov

    The lowest energy-binding conformation of an inhibitor bound to the dimeric interface of HIV-1 integrase core domain. The yellow region represents a unique allosteric binding site identified by affinity labeling and mass spectrometry and validated through mutagenesis. This site can provide a potential platform for the rational design of inhibitors selective for disruption of integrase multimerization.

  4. Single-molecule Imaging Analysis of Binding, Processive Movement, and Dissociation of Cellobiohydrolase Trichoderma reesei Cel6A and Its Domains on Crystalline Cellulose.

    PubMed

    Nakamura, Akihiko; Tasaki, Tomoyuki; Ishiwata, Daiki; Yamamoto, Mayuko; Okuni, Yasuko; Visootsat, Akasit; Maximilien, Morice; Noji, Hiroyuki; Uchiyama, Taku; Samejima, Masahiro; Igarashi, Kiyohiko; Iino, Ryota

    2016-10-21

    Trichoderma reesei Cel6A (TrCel6A) is a cellobiohydrolase that hydrolyzes crystalline cellulose into cellobiose. Here we directly observed the reaction cycle (binding, surface movement, and dissociation) of single-molecule intact TrCel6A, isolated catalytic domain (CD), cellulose-binding module (CBM), and CBM and linker (CBM-linker) on crystalline cellulose Iα The CBM-linker showed a binding rate constant almost half that of intact TrCel6A, whereas those of the CD and CBM were only one-tenth of intact TrCel6A. These results indicate that the glycosylated linker region largely contributes to initial binding on crystalline cellulose. After binding, all samples showed slow and fast dissociations, likely caused by the two different bound states due to the heterogeneity of cellulose surface. The CBM showed much higher specificity to the high affinity site than to the low affinity site, whereas the CD did not, suggesting that the CBM leads the CD to the hydrophobic surface of crystalline cellulose. On the cellulose surface, intact molecules showed slow processive movements (8.8 ± 5.5 nm/s) and fast diffusional movements (30-40 nm/s), whereas the CBM-Linker, CD, and a catalytically inactive full-length mutant showed only fast diffusional movements. These results suggest that both direct binding and surface diffusion contribute to searching of the hydrolysable point of cellulose chains. The duration time constant for the processive movement was 7.7 s, and processivity was estimated as 68 ± 42. Our results reveal the role of each domain in the elementary steps of the reaction cycle and provide the first direct evidence of the processive movement of TrCel6A on crystalline cellulose.

  5. HLA-DO increases bacterial superantigen binding to human MHC molecules by inhibiting dissociation of class II-associated invariant chain peptides.

    PubMed

    Pezeshki, Abdul Mohammad; Azar, Georges A; Mourad, Walid; Routy, Jean-Pierre; Boulassel, Mohamed-Rachid; Denzin, Lisa K; Thibodeau, Jacques

    2013-10-01

    HLA-DO (H2-O in mice) is an intracellular non-classical MHC class II molecule (MHCII). It forms a stable complex with HLA-DM (H2-M in mice) and shapes the MHC class II-associated peptide repertoire. Here, we tested the impact of HLA-DO and H2-O on the binding of superantigens (SAgs), which has been shown previously to be sensitive to the structural nature of the class II-bound peptides. We found that the binding of staphylococcal enterotoxin (SE) A and B, as well as toxic shock syndrome toxin 1 (TSST-1), was similar on the HLA-DO(+) human B cell lines 721.45 and its HLA-DO(-) counterpart. However, overexpressing HLA-DO in MHC class II(+) HeLa cells (HeLa-CIITA-DO) improved binding of SEA and TSST-1. Accordingly, knocking down HLA-DO expression using specific siRNAs decreased SEA and TSST-1 binding. We tested directly the impact of the class II-associated invariant chain peptide (CLIP), which dissociation from MHC class II molecules is inhibited by overexpressed HLA-DO. Loading of synthetic CLIP on HLA-DR(+) cells increased SEA and TSST-1 binding. Accordingly, knocking down HLA-DM had a similar effect. In mice, H2-O deficiency had no impact on SAgs binding to isolated splenocytes. Altogether, our results demonstrate that the sensitivity of SAgs to the MHCII-associated peptide has physiological basis and that the effect of HLA-DO on SEA and TSST-1 is mediated through the inhibition of CLIP release.

  6. Binding of atoms and stability of molecules in Hartree and Thomas-Fermi type theories. Part 4: Binding of neutral systems for the Hartree model

    SciTech Connect

    Catto, I.; Lions, P.L. )

    1993-01-01

    This paper is the fourth of a series devoted to the study of the stability of general molecular systems in Thomas-Fermi or Hartree type models. In the preceding part, the authors proved the binding of arbitrary neutral systems for Thomas-Fermi type theories and of planar neutral systems for the Hartree model. In this part, they manage to get rid of this restriction and thus prove the binding and the stability of arbitrary neutral systems for the Hartree model. 23 refs.

  7. Demonstration of specific binding of heparin to Plasmodium falciparum-infected vs. non-infected red blood cells by single-molecule force spectroscopy

    NASA Astrophysics Data System (ADS)

    Valle-Delgado, Juan José; Urbán, Patricia; Fernàndez-Busquets, Xavier

    2013-04-01

    Glycosaminoglycans (GAGs) play an important role in the sequestration of Plasmodium falciparum-infected red blood cells (pRBCs) in the microvascular endothelium of different tissues, as well as in the formation of small clusters (rosettes) between infected and non-infected red blood cells (RBCs). Both sequestration and rosetting have been recognized as characteristic events in severe malaria. Here we have used heparin and pRBCs infected by the 3D7 strain of P. falciparum as a model to study GAG-pRBC interactions. Fluorescence microscopy and fluorescence-assisted cell sorting assays have shown that exogenously added heparin has binding specificity for pRBCs (preferentially for those infected with late forms of the parasite) vs. RBCs. Heparin-pRBC adhesion has been probed by single-molecule force spectroscopy, obtaining an average binding force ranging between 28 and 46 pN depending on the loading rate. No significant binding of heparin to non-infected RBCs has been observed in control experiments. This work represents the first approach to quantitatively evaluate GAG-pRBC molecular interactions at the individual molecule level.Glycosaminoglycans (GAGs) play an important role in the sequestration of Plasmodium falciparum-infected red blood cells (pRBCs) in the microvascular endothelium of different tissues, as well as in the formation of small clusters (rosettes) between infected and non-infected red blood cells (RBCs). Both sequestration and rosetting have been recognized as characteristic events in severe malaria. Here we have used heparin and pRBCs infected by the 3D7 strain of P. falciparum as a model to study GAG-pRBC interactions. Fluorescence microscopy and fluorescence-assisted cell sorting assays have shown that exogenously added heparin has binding specificity for pRBCs (preferentially for those infected with late forms of the parasite) vs. RBCs. Heparin-pRBC adhesion has been probed by single-molecule force spectroscopy, obtaining an average binding force

  8. Molecularly imprinted protein recognition cavities bearing exchangeable binding sites for postimprinting site-directed introduction of reporter molecules for readout of binding events.

    PubMed

    Sunayama, Hirobumi; Takeuchi, Toshifumi

    2014-11-26

    Protein-imprinted cavities bearing exchangeable domains to be used for postimprinting fluorophore introduction to transform binding events into fluorescence changes were constructed in molecularly imprinted polymer (MIPs) matrixes prepared on glass substrates. Copolymerization was performed with acrylamide, N,N'-methylenebisaclylamide, and a newly designed functional group-exchangeable monomer, ({[2-(2-methacrylamido)ethyldithio]ethylcarbamoyl}methoxy)acetic acid (MDTA), in the presence of a model basic protein, lysozyme (Lyso); MDTA can interact with Lyso and assemble close to Lyso in the resulting polymer. After removal of Lyso, followed by a disulfide reduction to cleave the (ethylcarbamoylmethoxy)acetic acid moiety from the MDTA residues, the exposed thiol groups within the imprinted cavities were modified by aminoethylpyridyldisulfide to be transformed into aminoethyl groups that function as active sites for amine-reactive fluorophores. Fluorescein isothiocyanate (FITC) was then coupled with the aminoethyl groups, yielding site specifically FITC-modified signaling imprinted cavities for Lyso binding. Because the in-cavity fluorescent labeling was achieved via a disulfide linkage, it was easy to remove, exchange, and/or replace amine-reactive fluorophores. This facilitated the screening of fluorophores to select the highest readout for binding events, replace fluorophores when photobleaching occurred, and introduce other functions. The proposed molecular imprinting process, combined with postimprinting modifications, is expected to provide an affordable route to develop multifunctional MIPs for specific detection of protein binding events.

  9. New measurements of the sticking coefficient and binding energy of molecules on non-porous amorphous solid water in the submonolayer regime

    NASA Astrophysics Data System (ADS)

    He, Jiao; Acharyya, Kinsuk; Emtiaz, S. M.; Vidali, Gianfranco

    2016-06-01

    Sticking and adsorption of molecules on dust grains are two important processes in gas-grain interactions. We accurately measured both the sticking coefficient and the binding energy of several key molecules on the surface of amorphous solid water as a function of coverage.A time-resolved scattering technique was used to measure sticking coefficient of H2, D2, N2, O2, CO, CH4, and CO2 on non-porous amorphous solid water (np-ASW) in the low coverage limit over a wide range of surface temperatures. We found that the time-resolved scattering technique is advantageous over the conventional King-Wells method that underestimates the sticking coefficient. Based on the measured values we suggest a useful general formula of the sticking coefficient as a function of grain temperature and molecule-surface binding energy.We measured the binding energy of N2, CO, O2, CH4, and CO2 on np-ASW, and of N2 and CO on porous amorphous solid water (p-ASW). We were able to measure binding energies down to a fraction of 1% of a layer, thus making these measurements more appropriate for astrochemistry than the existing values. We found that CO2 forms clusters on np-ASW surface even at very low coverage; this may help in explaining the segregation of CO2 in ices. The binding energies of N2, CO, O2, and CH4 on np-ASW decrease with coverage in the submonolayer regime. Their values in the low coverage limit are much higher than what is commonly used in gas-grain models. An empirical formula was used to describe the coverage dependence of the binding energies. We used the newly determined binding energy distributions in a simulation of gas-grain chemistry for cold dense clouds and hot core models. We found that owing to the higher value of desorption energy in the sub-monlayer regime a fraction of all these ices stays much longer and to higher temperature on the grain surface compared to the case using single value energies as currently done in astrochemical models.This work was supported in

  10. Binding Characteristics of Small Molecules that Mimic Nucleocapsid Protein-induced Maturation of Stem-loop-1 of HIV-1 RNA†

    PubMed Central

    Chung, Janet; Ulyanov, Nikolai B.; Guilbert, Christophe; Mujeeb, Anwer; James, Thomas L.

    2010-01-01

    As a retrovirus, the human immunodeficiency virus (HIV-1) packages two copies of the RNA genome as a dimer in the infectious virion. Dimerization is initiated at the dimer initiation site (DIS) which encompasses stem-loop 1 (SL1) in the 5’-UTR of the genome. Study of genomic dimerization has been facilitated by the discovery that short RNA fragments containing SL1 can dimerize spontaneously without any protein factors. Based on the palindromic nature of SL1, a kissing loop model has been proposed. First, a metastable kissing dimer is formed via standard Watson-Crick base pairs and then converted into a more stable extended dimer by the viral nucleocapsid protein (NCp7). This dimer maturation in vitro is believed to mimic initial steps in the RNA maturation in vivo, which is correlated with viral infectivity. We previously discovered a small molecule activator, Lys-Ala-7-amido-4-methylcoumarin (KA-AMC), which facilitates dimer maturation in vitro, and determined aspects of its structure-activity relationship. In this report, we present measurements of the binding affinity of the activators and characterization of their interactions with the SL1 RNA. Guanidinium groups and increasing positive charge on the side chain enhance affinity and activity, but features in the aromatic ring at least partially decouple affinity from activity. Although KA-AMC can bind to multiple structural motifs, NMR study showed KA-AMC preferentially binds to unique structural motifs, such as the palindromic loop and the G-rich internal loop in the SL1 RNA. NCp7 binds to SL1 only an order of magnitude tighter than the best small molecule ligand tested. The study presented here provides guidelines for design of superior small molecule binders to the SL1 RNA that have the potential of being developed as an antiviral by either interfering with SL1-NCp7 interaction at the packaging and/or maturation stages. PMID:20565056

  11. Post-translational modification and conformational state of Heat Shock Protein 90 differentially affect binding of chemically diverse small molecule inhibitors

    PubMed Central

    Beebe, Kristin; Mollapour, Mehdi; Scroggins, Bradley; Prodromou, Chrisostomos; Xu, Wanping; Tokita, Mari; Taldone, Tony; Pullen, Lester; Zierer, Bettina K.; Lee, Min-Jung; Trepel, Jane; Buchner, Johannes; Bolon, Daniel; Chiosis, Gabriela; Neckers, Leonard

    2013-01-01

    Heat shock protein 90 (Hsp90) is an essential molecular chaperone in eukaryotes that facilitates the conformational maturation and function of a diverse protein clientele, including aberrant and/or over-expressed proteins that are involved in cancer growth and survival. A role for Hsp90 in supporting the protein homeostasis of cancer cells has buoyed interest in the utility of Hsp90 inhibitors as anti-cancer drugs. Despite the fact that all clinically evaluated Hsp90 inhibitors target an identical nucleotide-binding pocket in the N domain of the chaperone, the precise determinants that affect drug binding in the cellular environment remain unclear, and it is possible that chemically distinct inhibitors may not share similar binding preferences. Here we demonstrate that two chemically unrelated Hsp90 inhibitors, the benzoquinone ansamycin geldanamycin and the purine analog PU-H71, select for overlapping but not identical subpopulations of total cellular Hsp90, even though both inhibitors bind to an amino terminal nucleotide pocket and prevent N domain dimerization. Our data also suggest that PU-H71 is able to access a broader range of N domain undimerized Hsp90 conformations than is geldanamycin and is less affected by Hsp90 phosphorylation, consistent with its broader and more potent anti-tumor activity. A more complete understanding of the impact of the cellular milieu on small molecule inhibitor binding to Hsp90 should facilitate their more effective use in the clinic. PMID:23867252

  12. Specific Capture of Peptide-Receptive Major Histocompatibility Complex Class I Molecules by Antibody Micropatterns Allows for a Novel Peptide-Binding Assay in Live Cells.

    PubMed

    Dirscherl, Cindy; Palankar, Raghavendra; Delcea, Mihaela; Kolesnikova, Tatiana A; Springer, Sebastian

    2017-02-02

    Binding assays with fluorescently labeled ligands and recombinant receptor proteins are commonly performed in 2D arrays. But many cell surface receptors only function in their native membrane environment and/or in a specific conformation, such as they appear on the surface of live cells. Thus, receptors on live cells should be used for ligand binding assays. Here, it is shown that antibodies preprinted on a glass surface can be used to specifically array a peptide receptor of the immune system, i.e., the major histocompatibility complex class I molecule H-2K(b) , into a defined pattern on the surface of live cells. Monoclonal antibodies make it feasible to capture a distinct subpopulation of H-2K(b) and hold it at the cell surface. This patterned receptor enables a novel peptide-binding assay, in which the specific binding of a fluorescently labeled index peptide is visualized by microscopy. Measurements of ligand binding to captured cell surface receptors in defined confirmations apply to many problems in cell biology and thus represent a promising tool in the field of biosensors.

  13. Small Molecule Inhibition of Epstein - Barr Virus Nuclear Antigen-1 DNA Binding Activity Interferes with Replication and Persistence of the Viral Genome

    PubMed Central

    Noh, Ka-Won; Joo, Eun Hye; Zhao, Bo; Kieff, Elliott; Kang, Myung-Soo

    2014-01-01

    The replication and persistence of extra chromosomal Epstein-Barr virus (EBV) episome in latently infected cells are primarily dependent on the binding of EBV-encoded nuclear antigen 1 (EBNA1) to the cognate EBV oriP element. In continuation of the previous study, herein we characterized EBNA1 small molecule inhibitors (H20, H31) and their underlying inhibitory mechanisms. In silico docking analyses predicted that H20 fits into a pocket in the EBNA1 DNA binding domain (DBD). However, H20 did not significantly affect EBNA1 binding to its cognate sequence. A limited structure-relationship study of H20 identified a hydrophobic compound H31, as an EBNA1 inhibitor. An in vitro EBNA1 EMSA and in vivo EGFP-EBNA1 confocal microscopy analysis showed that H31 inhibited EBNA1-dependent oriP sequence-specific DNA binding activity, but not sequence-nonspecific chromosomal association. Consistent with this, H31 repressed the EBNA1-dependent transcription, replication, and persistence of an EBV oriP plasmid. Furthermore, H31 induced progressive loss of EBV episome. In addition, H31 selectively retarded the growth of EBV-infected LCL or Burkitt’s lymphoma cells. These data indicate that H31 inhibition of EBNA1-dependent DNA binding decreases transcription from and persistence of EBV episome in EBV-infected cells. These new compounds might be useful probes for dissecting EBNA1 functions in vitro and in vivo. PMID:24486954

  14. Cooperative binding of PhoB(DBD) to its cognate DNA sequence-a combined application of single-molecule and ensemble methods.

    PubMed

    Ritzefeld, Markus; Walhorn, Volker; Kleineberg, Christin; Bieker, Adeline; Kock, Klaus; Herrmann, Christian; Anselmetti, Dario; Sewald, Norbert

    2013-11-19

    A combined approach based on isothermal titration calorimetry (