Identification of a thermo-regulated glutamine-binding protein from Yersinia pestis.

Here we present modeling and NMR spectroscopic evidence that the function of a Yersinia pestis pMT1 plasmid protein, designated as orf38, is most likely a glutamine binding protein. The modeling was homology-based at a very low level of sequence identity ( approximately 16%) and involved structural comparison of multiple templates, as well as template-substrate interaction analyses. Transferred nuclear Overhauser and saturation transfer difference experiments were used to characterize the binding of sugars and amino acids to orf38.

Selective high-affinity ligand antibody mimics for cancer diagnosis and therapy: initial application to lymphoma/leukemia.

Purpose: More than two decades of research and clinical trials have shown radioimmunotherapy to be a promising approach for treating various forms of cancer. Lym-1 antibody, which binds selectively to HLA-DR10 on malignant B-cell lymphocytes, has proved to be effective in delivering radionuclides to non-Hodgkin's lymphoma and leukemia. Using a new approach to create small synthetic molecules that mimic the targeting properties of the Lym-1 antibody, a prototype, selective high-affinity ligand (SHAL), has been developed to bind to a unique region located within the Lym-1 epitope on HLA-DR10.

Recombinant expression of the beta-subunit of HLA-DR10 for the selection of novel lymphoma targeting molecules.

Selective high-affinity ligands (SHALs) were selected as substitutes for monoclonal antibodies (mAbs) to deliver radioisotopes to malignant tumors. Because a SHAL (5 KD) is considerably smaller in comparison to an antibody (150 KD), a significant therapeutic index (TI) enhancement for radioimmunotherapy (RIT) is anticipated. The antibody-antigen (Ab-Ag) model system chosen for the development of SHALs consists of Lym-1, a MAb with proven selectivity in non-Hodgkin's lymphoma (NHL) patients and its well-characterized Ag, the beta subunit of HLA DR10.

Characteristics of dimeric (bis) bidentate selective high affinity ligands as HLA-DR10 beta antibody mimics targeting non-Hodgkin's lymphoma.

Despite their large size, antibodies have proven to be suitable radioisotope carriers to deliver systemic radiotherapy, often molecular image-based, for lymphoma and leukemia. To mimic antibody (Ab) targeting behavior while decreasing size by 50-100x, a combination of computational and experimental methods were used to generate molecules that bind to unique sites within the HLA-DR epitopic region of Lym-1, an Ab shown effective in patients. Lym-1 Ab mimics (synthetic high afinity ligands; SHALs) were generated and studied in vitro, using live cell binding assays, and/or pharmacokinetic studies over 24 h in xenografted mice given 1 or 20 microg SHAL doses i.

Pharmacokinetic characterization in xenografted mice of a series of first-generation mimics for HLA-DR antibody, Lym-1, as carrier molecules to image and treat lymphoma.

Unlabelled: Despite their large size, antibodies (Abs) are suitable carriers to deliver systemic radiotherapy, often molecular image-based, for lymphoma and leukemia. Lym-1 Ab has proven to be an effective radioisotope carrier, even in small amounts, for targeting human leukocyte antigen DR (HLA-DR), a surface membrane protein overexpressed on B-cell lymphoma. Pairs of molecules (referred to as ligands), shown by computational and experimental methods to bind to each of 2 sites within the Lym-1 epitopic region, have been linked to generate small (<2 kDa) molecules (referred to as selective high-affinity ligands [SHALs]) to mimic the targeting properties of Lym-1 Ab.

Monitoring dynamic protein expression in living E. coli. Bacterial cells by laser tweezers Raman spectroscopy.

Background: Laser tweezers Raman spectroscopy (LTRS) is a novel, nondestructive, and label-free method that can be used to quantitatively measure changes in cellular activity in single living cells. Here, we demonstrate its use to monitor changes in a population of E. coli cells that occur during overexpression of a protein, the extracellular domain of myelin oligodendrocyte glycoprotein [MOG(1-120)].

Direct antilymphoma activity of novel, first-generation "antibody mimics" that bind HLA-DR10-positive non-Hodgkin's lymphoma cells.

A first-generation series of novel small molecules, collectively known as selective high-affinity ligands (SHALs), were designed and synthesized to mimic the binding of Lym-1, a monoclonal antibody (mAb) shown to be an effective cytotoxic and radionuclide carrier molecule for targeting non-Hodgkin's lymphoma (NHL). Created as radionuclide targeting molecules, these SHALs were intended to have the human leukocyte antigen-DR (HLA-DR) selectivity of Lym-1 mAb and the pharmacokinetics of a small molecule. Because of the remarkable bioactivity of Lym-1 in vitro, the direct antilymphoma activity of three of these SHALs was tested.

PhIP carcinogenicity in breast cancer: computational and experimental evidence for competitive interactions with human estrogen receptor.

Many carcinogens have been shown to cause tissue specific tumors in animal models. The mechanism for this specificity has not been fully elucidated and is usually attributed to differences in organ metabolism. For heterocyclic amines, potent carcinogens that are formed in well-done meat, the ability to either bind to the estrogen receptor and activate or inhibit an estrogenic response will have a major impact on carcinogenicity.

Application of NMR methods to identify detection reagents for use in development of robust nanosensors.

Nuclear magnetic resonance (NMR) spectroscopy is a powerful technique for studying bimolecular interactions at the atomic scale. Our NMR laboratory is involved in the identification of small molecules, or ligands, that bind to target protein receptors such as tetanus neurotoxin (TeNT) and botulinum neurotoxin, anthrax proteins, and HLA-DR10 receptors on non-Hodgkin lymphoma cancer cells. Once low-affinity binders are identified, they can be linked together to produce multidentate synthetic high-affinity ligands (SHALs) that have very high specificity for their target protein receptors.

Solution structure of an O6-[4-oxo-4-(3-pyridyl)butyl]guanine adduct in an 11 mer DNA duplex: evidence for formation of a base triplex.

The pyridyloxobutylating agents derived from metabolically activated tobacco-specific nitrosamines can covalently modify guanine bases in DNA at the O(6) position. The adduct formed, O(6)-[4-oxo-4-(3-pyridyl)butyl]guanine ([POB]dG), results in mutations that can lead to tumor formation, posing a significant cancer risk to humans exposed to tobacco smoke. A combined NMR-molecular mechanics computational approach was used to determine the solution structure of the [POB]dG adduct within an 11mer duplex sequence d(CCATAT-[POB]G-GCCC).

Extracellular domain of myelin oligodendrocyte glycoprotein (MOG) exhibits solvent-dependent conformational transitions.

The conformation of the non-glycosylated recombinant form of the extracellar domain of rat MOG (rMOG(1-125)) dissolved in different solvent conditions was studied by CD spectroscopy. The results show that rMOG(1-125) exhibits a predominantly beta sheet conformation in aqueous buffer solution at pH 7.5 and that this 'beta-form' is stabilized by zwitterionic phospholipids, DPC and LPCP.

An empirical correlation between secondary structure content and averaged chemical shifts in proteins.

It is shown that the averaged chemical shift (ACS) of a particular nucleus in the protein backbone empirically correlates well to its secondary structure content (SSC). Chemical shift values of more than 200 proteins obtained from the Biological Magnetic Resonance Bank are used to calculate ACS values, and the SSC is estimated from the corresponding three-dimensional coordinates obtained from the Protein Data Bank. ACS values of (1)H(alpha) show the highest correlation to helical and sheet structure content (correlation coefficient of 0.

Identification of novel small molecules that bind to two different sites on the surface of tetanus toxin C fragment.

A combination of computational methods, electrospray ionization mass spectroscopy (ESI-MS), and NMR spectroscopy has been used to identify novel small molecules that bind to two adjacent sites on the surface of the C fragment of tetanus toxin (TetC). One of these sites, Site-1, binds gangliosides present on the surface of motor neurons, while Site-2 is a highly conserved deep cleft in the structures of the tetanus (TeNT) and botulinum (BoNT) neurotoxins. ESI-MS was used to experimentally determine which of the top 11 computationally predicted Site-2 candidates bind to TetC.

Marmoset fine B cell and T cell epitope specificities mapped onto a homology model of the extracellular domain of human myelin oligodendrocyte glycoprotein.

Aberrant association of autoantibodies with myelin oligodendrocyte glycoprotein (MOG), an integral membrane protein of the central nervous system (CNS) myelin, has been implicated in the pathogenesis of multiple sclerosis (MS). Sensitization of nonhuman primates (Callithrix jacchus marmosets) against the nonglycosylated, recombinant N-terminal domain of rat MOG (residues 1-125) reproduces an MS-like disease in which MOG-specific autoantibodies directly mediate demyelination. To assess the interrelationship between MOG structure and the induction of autoimmune CNS diseases and to enable structure-based rational design of therapeutics, a homology model of human MOG(2-120) was constructed based on consensus residues found in immunoglobulin superfamily variable-type proteins having known structures.