Biomedical subjects

D Kuchnir Fygenson

Publications and source records attributed to D Kuchnir Fygenson.

5 recordsLinked to original sources

PubMed2004

Tau induces cooperative Taxol binding to microtubules.

Taxol and tau are two ligands that stabilize the microtubule (MT) lattice. Taxol is an anti-mitotic drug that binds beta tubulin in the MT interior. Tau is a MT-associated protein that binds both alpha and beta tubulin on the MT exterior. Both Taxol and tau reduce MT dynamics and promote tubulin polymerization. Tau alone also acts to bundle, stiffen, and space MTs. A structural study recently suggested that Taxol and tau may interact by binding to the same site. Using fluorescence recovery after photobleaching, we find that tau induces Taxol to bind MTs cooperatively depending on the tau concentration. We develop a model that correctly fits the data in the absence of tau, yields the equilibrium dissociation constant of approximately 2 microM, and determines the escape rate of Taxol through one pore to be 1.7 x 10(3) (M x s)(-1). Extension of the model yields a measure of Taxol cooperativity with a Hill coefficient of at least 15 when tau is present at a 1:1 molar ratio with tubulin.

Animals↗

PubMed2004

Variability-based sequence alignment identifies residues responsible for functional differences in alpha and beta tubulin.

alpha and beta Tubulin are well-characterized paralogs with similar structures and functions. We quantify the variability of every amino acid position in both tubulins from the aligned sequences of their numerous known orthologs. By aligning the variability profiles, we identify residues that differ significantly in variability between alpha and beta tubulin. Most of these residues are part of well-defined secondary structures and are clustered around the nucleotide binding pocket, the site of greatest functional difference between the two paralogs. The remaining residues of large difference in variability are located in the N-terminal loop between H1 and S2. We therefore predict that certain residues in this unstructured region also contribute to a functional difference between alpha and beta tubulin. Furthermore, we find the most restrictive variability-based alignment is nearly identical to the true structure-based alignment. Thus, by using a stringent variability-based alignment to approximate the true alignment, the method introduced here may predict sites of functional distinction between paralogous proteins even in the absence of structural information.

Algorithms↗

PubMed2003

Size exclusion and diffusion of fluoresceinated probes within collagen fibrils.

The diffusion of fluoresceinated probes inside single collagen fibrils was investigated by imaging the migration of fluorescence along the fibrils in oil and by monitoring fluorescence recovery after photobleaching (FRAP). Probes were excluded from the fibrils according to their size. Probes that were not excluded diffused in the fibrils, but FRAP occurred 6 x 10(-4) times more slowly than in water due to binding interactions between collagen and the probes. The dissociation constant of the fluorescein-collagen complex was determined (K(D)=1.8+/-0.1 microM).

Journal Article↗

PubMed2003

Mobility of taxol in microtubule bundles.

Mobility of taxol inside microtubules was investigated using fluorescence recovery after photobleaching on flow-aligned bundles. Bundles were made of microtubules with either GMPCPP or GTP at the exchangeable site on the tubulin dimer. Recovery times were sensitive to bundle thickness and packing, indicating that taxol molecules are able to move laterally through the bundle. The density of open binding sites along a microtubule was varied by controlling the concentration of taxol in solution for GMPCPP samples. With >63% sites occupied, recovery times were independent of taxol concentration and, therefore, inversely proportional to the microscopic dissociation rate, k(off). It was found that 10k(off)(GMPCPP) approximately equal k(off)(GTP), consistent with, but not fully accounting for, the difference in equilibrium constants for taxol on GMPCPP and GTP microtubules. With <63% sites occupied, recovery times decreased as approximately [Tax](-1/5) for both types of microtubules. We conclude that the diffusion of taxol inside the microtubule bundle is hindered by rebinding events when open sites are within approximately 7 nm of each other.

Binding Sites↗

PubMed2002

DNA melting in the presence of fluorescent intercalating oxazole yellow dyes measured with a gel-based assay.

We measured the effect of the intercalating oxazole yellow DNA dye quinolinium,4-[(3-methyl-2(3H)-benzoxazolylidene)methyl]-1-[3-(trimethylammonio)propyl]-,diiodide (YO-PRO) and its homodimer (YOYO) on the melting of self-complementary DNA duplexes using a gel-based assay. The assay, which requires a self-complementary DNA sequence, is independent of the optical properties of the molecules in solution. The melting temperature of the DNA is observed to increase in direct proportion to the number of occupied intercalation sites on the DNA, irrespective of whether the dye molecules are in monomer or dimer form. The increase is approximately 2.5 degrees C for each intercalation site occupied in the presence of 38 mM [Na(+)], for dye/duplex ratios in which less than 1/5 of the available intercalation sites are occupied.

Benzoxazoles↗