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Ivan J Dmochowski

Publications and source records attributed to Ivan J Dmochowski.

10 recordsLinked to original sources
PubMed2006

Designing 129Xe NMR biosensors for matrix metalloproteinase detection.

Xenon-129 biosensors offer an attractive alternative to conventional MRI contrast agents due to the chemical shift sensitivity and large nuclear magnetic signal of hyperpolarized (129)Xe. Here, we report the first enzyme-responsive (129)Xe NMR biosensor. This compound was synthesized in 13 steps by attaching the consensus peptide substrate for matrix metalloproteinase-7 (MMP-7), an enzyme that is upregulated in many cancers, to the xenon-binding organic cage, cryptophane-A. The final coupling step was achieved on solid support in 80-92% yield via a copper (I)-catalyzed [3+2] cycloaddition. In vitro enzymatic cleavage assays were monitored by HPLC and fluorescence spectroscopy. The biosensor was determined to be an excellent substrate for MMP-7 (K(M) = 43 microM, V(max) = 1.3 x 10(-)(8) M s(-1), k(cat)/K(M) = 7,200 M(-1) s(-1)). Enzymatic cleavage of the tryptophan-containing peptide led to a dramatic decrease in Trp fluorescence, lambda(max) = 358 nm. Stern-Volmer analysis gave an association constant of 9000 +/- 1,000 M(-1) at 298 K between the cage and Trp-containing hexapeptide under enzymatic assay conditions. Most promisingly, (129)Xe NMR spectroscopy distinguished between the intact and cleaved biosensors with a 0.5 ppm difference in chemical shift. This difference most likely reflected a change in the electrostatic environment of (129)Xe, caused by the cleavage of three positively charged residues from the C-terminus. This work provides guidelines for the design and application of new enzyme-responsive (129)Xe NMR biosensors.

Amino Acid Sequence↗
PubMed2006

Design of functional ferritin-like proteins with hydrophobic cavities.

Ferritin four-helix bundle subunits self-assemble to create a stable multimer with a large central hydrophilic cavity where metal ions bind. To explore the versatility of this reaction vessel, computational design was used to generate cavities with increasingly apolar surface areas inside a dodecameric ferritin-like protein, Dps. Cavity mutants, in which as many as 120 surface accessible hydrophilic residues were replaced with hydrophobic amino acids, were shown to still assemble properly using size-exclusion chromatography and dynamic light scattering measurements. Wild-type Dps exhibited highly cooperative subunit folding and assembly, which was monitored by changes in Trp fluorescence and UV circular dichroism. The hydrophobic cavity mutants showed distinctly less cooperative unfolding behavior, with one mutant forming a partially assembled intermediate upon guanidine denaturation. Although the stability of Dps to such denaturation decreased with increasing apolar surface area, all proteins exhibited high melting temperatures, T(m) = 74-90 degrees C. Despite the large number of mutations, near-native ability to mineralize iron was maintained. This work illustrates the versatility of the ferritin scaffold for engineering large protein cavities with novel properties.

Bacterial Proteins↗
PubMed2006

Specific delivery of corroles to cells via noncovalent conjugates with viral proteins.

PURPOSE: Corroles are amphiphilic macrocycles that can bind and transport metal ions, and thus may be toxic to cells. We predicted that anionic corroles would poorly enter cells due to the negatively charged cell membrane, but could be ideal tumor-targeted drugs if appropriate carriers enabled delivery into tumor cells. In this work, we test the hypothesis that recombinant cell penetrating proteins of the adenovirus (Ad) capsid form noncovalent conjugates with corroles to facilitate target-specific delivery and cell death. METHODS: Corroles mixed with recombinant proteins were tested for conjugate assembly, cell penetration, stability, targeted binding, and cell killing in vitro. RESULTS: Sulfonated corroles entered cells only with carrier proteins, and formed stable complexes with recombinant Ad capsid proteins. ErbB receptor-targeted conjugates were cytotoxic to ErbB2-positive but not ErbB2-negative breast cancer cells, whereas molar equivalents of free corrole had no effect on these cells. CONCLUSIONS: Sulfonated corroles are cytotoxic to ErbB2-positive breast cancer cells when delivered by a targeted cell penetrating protein. The relatively low dose required to accomplish this compared to untargeted compounds suggests that corroles may lend themselves to targeted therapy. Importantly, the amphiphilicity of corroles enables a unique approach to bioconjugate formation whereby the carrier and drug form a stable complex by noncovalent assembly.

Cell Death↗
PubMed2005

Photoregulation of DNA polymerase I (Klenow) with caged fluorescent oligodeoxynucleotides.

The DNA polymerase reaction by Klenow fragment (KF) was efficiently regulated with UV light using a 25-mer caged fluorescent oligodeoxynucleotide (CFO) as the template. The CFO was functionalized with a fluorescein reporter (Fl) and photocleavable DABSYL quencher moiety (Dab). With Fl and Dab at adjacent cytidines in the middle at the template, KF was blocked from extending a complementary 12-mer primer. Upon UV photolysis of the DABSYL blocking group under aerobic conditions, fluorescein emission was restored and 50% of the primers were fully extended by KF.

DNA Polymerase I↗
PubMed2005

Phototriggering of caged fluorescent oligodeoxynucleotides.

[Structure: see text] We describe a synthetic route for incorporating a photocleavable (PC) DABSYL moiety and fluorescein at adjacent cytidines in the middle of a 25-mer oligodeoxynucleotide. In hybridization studies, both fluorescein and the photocleavable DABSYL reduced the Tm about 6 degrees C separately. UV irradiation (355 nm, 2 min) removed the PC-DABSYL moiety, which increased the fluorescence intensity 51-fold and restored the melting temperature. Caged fluorescent oligodeoxynucleotides (CFOs) will allow many DNA processes to be controlled with light.

Base Sequence↗
PubMed2004

Using reporter genes to study cis-regulatory elements.

This chapter summarizes four powerful assays for analyzing gene expression in cis-regulatory studies. The enzymatic assays (CAT, luciferase, lacZ) are currently limited by their application to embryo homogenates or fixed samples, but offer more robust analysis of gene activity than GFP. Assays based on CAT enzymatic activity or on CAT mRNA detection by WMISH are laborious but are well established for accurately quantifying gene expression and to determine spatial patterns at defined timepoints during development. LacZ assays are the current standard for spatially visualizing gene products in whole-mount fixed embryos. They are very sensitive but they provide limited temporal or quantitative information due to the perdurance of beta-galactosidase and the subtleties of the staining technique. Recently developed luciferase assays promise to be even more sensitive and accurate than the CAT and lacZ assays, and applicable to living cells and embryos. But, they have not yet been well established in invertebrate deuterostome research. GFP allows visualization of gene expression within living embryos. But because this is not an enzymatic assay, sensitivity can be a problem, particularly for weak promoters. Furthermore, imaging live embryos and quantifying gene expression in space and time (due to scattering of light by tissue, the perdurance of GFP, and other experimental details) is currently fraught with challenges. Ongoing improvements in imaging technology and the advent of multiple fluorescent proteins, as well as fluorescent and luminescent assays for vital imaging, will dramatically facilitate studies of gene expression in the coming decade.

Animals↗
PubMed2003

Nanosecond photoreduction of cytochrome p450cam by channel-specific Ru-diimine electron tunneling wires.

We report the synthesis and characterization of Ru-diimine complexes designed to bind to cytochrome p450cam (CYP101). The sensitizer core has the structure [Ru(L(2))L'](2+), where L' is a perfluorinated biphenyl bridge (F(8)bp) connecting 4,4'-dimethylbipyridine to an enzyme substrate (adamantane, F(8)bp-Ad), a heme ligand (imidazole, F(8)bp-Im), or F (F(9)bp). The electron-transfer (ET) driving force (-deltaG degrees ) is varied by replacing the ancillary 2,2'-bipyridine ligands with 4,4',5,5'-tetramethylbipyridine (tmRu). The four complexes all bind p450cam tightly: Ru-F(8)bp-Ad (1, K(d) = 0.077 microM); Ru-F(8)bp-Im (2, K(d) = 3.7 microM); tmRu-F(9)bp (3, K(d) = 2.1 microM); and tmRu-F(8)bp-Im (4, K(d) = 0.48 microM). Binding is predominantly driven by hydrophobic interactions between the Ru-diimine wires and the substrate access channel. With Ru-F(8)bp wires, redox reactions can be triggered on the nanosecond time scale. Ru-wire 2, which ligates the heme iron, shows a small amount of transient heme photoreduction (ca. 30%), whereas the transient photoreduction yield for 4 is 76%. Forward ET with 4 occurs in roughly 40 ns (k(f) = 2.8 x 10(7) s(-)(1)), and back ET (Fe(II) --> Ru(III), k(b) approximately 1.7 x 10(8) s(-)(1)) is near the coupling-limited rate (k(max)). Direct photoreduction was not observed for 1 or 3. The large variation in ET rates among the Ru-diimine:p450 conjugates strongly supports a through-bond model of Ru-heme electronic coupling.

Camphor 5-Monooxygenase↗
PubMed2002

Quantitative imaging of cis-regulatory reporters in living embryos.

A confocal laser scanning microscopy method has been developed for the quantitation of green fluorescent protein (GFP) as a reporter of gene activity in living three-dimensional structures such as sea urchin and starfish embryos. This method is between 2 and 50 times more accurate than conventional confocal microscopy procedures depending on the localization of GFP within an embryo. By using coinjected Texas red dextran as an internal fluorescent standard, the observed GFP intensity is corrected for variations in laser excitation and fluorescence collection efficiency. To relate the recorded image intensity to the number of GFP molecules, the embryos were lysed gently, and a fluorometric analysis of their contents was performed. Confocal laser scanning microscopy data collection from a single sea urchin blastula required less than 2 min, thereby allowing gene expression in dozens of embryos to be monitored in parallel with high spatial and temporal resolution.

Animals↗