D. Fygenson

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Papers by D. Fygenson

Tau induces cooperative Taxol binding to microtubules

Proceedings of the National Academy of Sciences, 2004

Taxol and tau are two ligands that stabilize the microtubule (MT) lattice. Taxol is an anti-mitot... more Taxol and tau are two ligands that stabilize the microtubule (MT) lattice. Taxol is an anti-mitotic drug that binds β tubulin in the MT interior. Tau is a MT-associated protein that binds both α and β 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 ≈2 μM, and determines the escape rate of Taxol through one pore to be 1.7 × 10 3 (M·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.

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Remotification of Five Introductory Modern Physics labs for ~$1k apiece

The pandemic forced educators of instructional physics lab classes to rethink how labs are taught... more The pandemic forced educators of instructional physics lab classes to rethink how labs are taught. We targeted introductory modern physics labs for remotification. When exploring the photo-electric effect, diffraction and interference, gamma-ray absorption, atomic spectra, and the Franck-Hertz experiment, which all involve sophisticated equipment, students engage primarily by pressing buttons, turning knobs, and reading displays. We were able to adapt and motorize classroom equipment for nearly equivalent remote engagement at relatively low cost. This was possible using consumer-grade technology that has matured over the last 15 years, namely 3D printing, single-board computers, webcams, and open-source programming libraries. In this presentation, we will demonstrate how students view and control our apparatus through a website and discuss elements and principles that have been key for successful deployment and student learning. Such remotified experiments may have post-pandemic rel...

Mobility of taxol in microtubule bundles

Mobility of taxol inside microtubules was investigated using fluorescence recovery after photoble... more 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, koff. It was found that 10 k GMPCPP off! kGTPoff; 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;[Tax]"1/5 for both types of microtubules. We conclude that the diffusion of taxol in...

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Letter Hairpins with Poly-C Loops Stabilize Four Types of Fluorescent Ag

Self-assembly of precisely defined DNA nanotube superstructures using DNA origami seeds

Nanoscale, Jan 5, 2017

We demonstrate a versatile process for assembling micron-scale filament architectures by controll... more We demonstrate a versatile process for assembling micron-scale filament architectures by controlling where DNA tile nanotubes nucleate on DNA origami assemblies. "Nunchucks," potential mechanical magnifiers of nanoscale dynamics consisting of two nanotubes connected by a dsDNA linker, form at yields sufficient for application and consistent with models.

format_quoteNunchuck seeds measured 64 by 21 nm with a linker length of 27.9 ± 2.9 nm, confirming design predictions.format_quote

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Spectral studies of optical coherence in DNA-encapsulated silver clusters

Novel emitters consisting of silver nanoclusters that are bound to single-stranded DNA show great... more

Amino acid variability in tubulin

ABSTRACT

The Effects of Concentration and Temperature on Vesicle Adsorption and Bilayer Formation

ABSTRACT

Microtubules and vesicles under controlled tension

Physical Review E, 1997

Microtubules trapped inside cell-sized vesicles ͑ϳ10-m diameter͒ define shapes reminiscent of liv... more Microtubules trapped inside cell-sized vesicles ͑ϳ10-m diameter͒ define shapes reminiscent of living cells. In this work, three configurations are studied: an individual microtubule, a linear bundle, and a circular band. Mechanical properties are probed by varying the vesicle membrane tension using micropipet aspiration. Quantitative results include measurements of the persistence length of a single microtubule, 6.3Ϯ2.4 mm, and the stretching modulus of the vesicle membrane, k s у140 erg/cm 2 . The number of microtubules in a typical bundle is ϳ100, and the total length in a typical band is ϳ0.5 cm. A tension-induced first-order transition from linear bundle to circular band is observed and modeled. The loss of stability of the bent bundle is associated with a saddle-node bifurcation.

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Spontaneous nucleation of microtubules

Physical review. E, Statistical physics, plasmas, fluids, and related interdisciplinary topics, 1995

The spontaneous nucleation of individual microtubules from tubulin dimers is observed directly un... more The spontaneous nucleation of individual microtubules from tubulin dimers is observed directly under conditions of marginal nucleation. The results are compared with measurements of bulk turbidity following a deep quench [W. A. Voter and H. P. Erickson, J. Biol. Chem. 259, 10 430 (1984)]. The size of the critical microtubule nucleus is 12+/-2 tubulin dimers in the two regimes, but

DNA polymerase fidelity: from genetics toward a biochemical understanding

Genetics, 1998

This review summarizes mutagenesis studies, emphasizing the use of bacteriophage T4 mutator and a... more This review summarizes mutagenesis studies, emphasizing the use of bacteriophage T4 mutator and antimutator strains. Early genetic studies on T4 identified mutator and antimutator variants of DNA polymerase that, in turn, stimulated the development of model systems for the study of DNA polymerase fidelity in vitro. Later enzymatic studies using purified T4 mutator and antimutator polymerases were essential in elucidating mechanisms of base selection and exonuclease proofreading. In both cases, the base analogue 2-aminopurine (2AP) proved tremendously useful-first as a mutagen in vivo and then as a probe of DNA polymerase fidelity in vitro. Investigations into mechanisms of DNA polymerase fidelity inspired theoretical models that, in turn, called for kinetic and thermodynamic analyses. Thus, the field of DNA synthesis fidelity has grown from many directions: genetics, enzymology, kinetics, physical biochemistry, and thermodynamics, and today the interplay continues. The relative cont...

format_quoteVariation in DNA polymerase background contributed up to 10^4-fold differences in synthesis accuracy according to different polymerase conditions.format_quote

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Phase diagram of microtubules

Physical Review E, 1994

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Size exclusion and diffusion of fluoresceinated probes within collagen fibrils

Physical Review E, 2003

The diffusion of fluoresceinated probes inside single collagen fibrils was investigated by imagin... more 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 ͑FRAP͒. Probes were excluded from the fibrils according to their size. Probes that were not excluded diffused in the fibrils, but FRAP occurred 6ϫ10 Ϫ4 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 ϭ1.8Ϯ0.1 M).

format_quoteHigher molecular weight dextrans (10 kD) are excluded from collagen fibrils based solely on size, emphasizing selective transport in biological matrices.format_quote

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Effects of 3-repeat tau on taxol mobility through microtubules

Both the anti-cancer drug taxol and the microtubule-associated protein tau suppress dynamics of m... more Both the anti-cancer drug taxol and the microtubule-associated protein tau suppress dynamics of microtubules (MT). We have observed taxol mobility with full-length 3-repeat tau, one of six tau isoforms, using fluorescence recovery after photobleaching (FRAP) on MTs and compare with earlier results on recombinant full-length adult 4-repeat tau. Taxol mobility becomes highly sensitive to taxol concentration in the presence of

Multimerization of DNA Origami Structures in Two Dimensions

ABSTRACT DNA nanotechnology, here in particular DNA origami, is based on self-assembly and can be... more ABSTRACT DNA nanotechnology, here in particular DNA origami, is based on self-assembly and can be used to construct arbitrary three-dimensional structures with nanometer precision. The dimensions of such DNA origami structures are typically on the order of a hundred nanometers or smaller. To achieve large-scale two-dimensional lattices that could be employed as scaffolds for crystalline arrangement of biomolecules and proteins, individual DNA origami tiles need to be assembled hierarchically. We work on the multimerization of DNA origami structures into extended one- and two- dimensional lattices that can cover areas of several square micrometers. This is achieved by complementary single stranded DNAs (sticky ends) at specific positions on the DNA origami objects that we intend to grow into periodic structures. We study the effect of varying multimerization conditions such as annealing temperatures, length of sticky ends and salt concentration on the quality and size of the resulting lattice.

THE EFFECTS OF b, g COMPLEX PROCESSIVITY PROTEINS AND e PROOFREADING EXONUCLEASE ON NUCLEOTIDE MISINCORPORATION EFFICIENCIES

The fidelity of Escherichia coli DNA polymerase III (pol III) is measured and the effects of ␤, ␥... more The fidelity of Escherichia coli DNA polymerase III (pol III) is measured and the effects of ␤, ␥ processivity and ⑀ proofreading subunits are evaluated using a gel kinetic assay. Pol III holoenzyme synthesizes DNA with extremely high fidelity, misincorporating dTMP, dAMP, and dGMP opposite a template G target with efficiencies f inc ‫؍‬ 5.6 ؋ 10 ؊6 , 4.2 ؋ 10 ؊7 , and 7 ؋ 10 ؊7 , respectively. Elevated dGMP⅐G and dTMP⅐G misincorporation efficiencies of 3.2 ؋ 10 ؊5 and 5.8 ؋ 10 ؊4 , attributed to a "dNTP-stabilized" DNA misalignment mechanism, occur when C and A, respectively, are located one base downstream from the template target G. At least 92% of misinserted nucleotides are excised by pol III holoenzyme in the absence of a next correct "rescue" nucleotide. As rescue dNTP concentrations are increased, pol III holoenzyme suffers a maximum 8-fold reduction in fidelity as proofreading of mispaired primer termini are reduced in competition with incorporation of a next correct nucleotide. Compared with pol III holoenzyme, the ␣ holoenzyme, which cannot proofread, has 47-, 32-, and 13-fold higher misincorporation rates for dGMP⅐G, dTMP⅐G, and dAMP⅐G mispairs. Both the ␤, ␥ complex and the downstream nucleotide have little effect on the fidelity of catalytic ␣ subunit. An analysis of the gel kinetic fidelity assay when multiple polymerase-DNA encounters occur is presented in the "Appendix" (see Fygenson, D. K., and Goodman, M. F. (1997) J. Biol. Chem. 272, 27931-27935 (accompanying paper)).

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Fluorescent silver nanocluster DNA probes for multiplexed detection using microfluidic capillary electrophoresis

The Analyst, 2015

We tune the electrophoretic mobilities of silver nanocluster DNA probes for Hepatitis A, B and C ... more

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Active DNA gels

Research into the mechanics and fluctuations of living cells has revealed the key role played by ... more Research into the mechanics and fluctuations of living cells has revealed the key role played by the cytoskeleton, a gel of stiff filaments driven out of equilibrium by force-generating motor proteins. Inspired by the extraordinary mechanical functions that the cytoskeleton imparts to the cell, we sought to create an artificial gel with similar characteristics. We identified DNA, and DNA-based motor proteins, as functional counterparts to the constituents of the cytoskeleton. We used DNA selfassembly to create a gel, and characterized its fluctuations and mechanics both before and after activation by the motor. We found that certain aspects of the DNA gel quantitatively match those of cytoskeletal networks, indicating the universal features of motor-driven, non-equilibrium networks.

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Hairpins with Poly-C Loops Stabilize Four Types of Fluorescent Ag n :DNA

The Journal of Physical Chemistry C, 2009

Supplemental Materials 1. Quantum yield estimates for 9C and 7C hairpins 2. Quantification of flu... more Supplemental Materials 1. Quantum yield estimates for 9C and 7C hairpins 2. Quantification of fluorescence intensities for 9C time series 3. Quantification of mass abundances for 9C time series Relative quantum yields (compared to fluorescein pH 7.5) 9C hairpin (green): 3.7% 9C hairpin (red): N.A. 7C hairpin (green): N.A. 7C hairpin (red): 34% Estimated absolute quantum yields 9C hairpin (green): ~3% 7C hairpin (red): ~30%

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UV Excitation of DNA Stabilized Ag Cluster Fluorescence via the DNA Bases

The Journal of Physical Chemistry C, 2011

ABSTRACT We report UV excitation through the DNA bases as a universal pathway to visible emission... more ABSTRACT We report UV excitation through the DNA bases as a universal pathway to visible emission from DNA stabilized silver cluster fluorophores (AgDNAs). AgDNAs with peak emissions throughout the visible spectrum all exhibit two peaks in fluorescence excitation: a visible peak at a wavelength 50–150 nm shorter than the emission peak and a UV peak between 260 and 270 nm depending on the specifics of the DNA strand. UV excitation produces the same emission spectrum as visible excitation with 2–4 times greater intensity depending on the specific emitter. The excellent agreement between AgDNA excitation and absorption spectra, the similarity between AgDNA and bare DNA absorption spectra, and the ubiquity of UV excitation for all the AgDNAs studied strongly suggest that UV excitation proceeds via the DNA bases rather than by direct excitation of higher energy excited states of the Ag cluster. We explore the possibility that UV excitation can provide information about the type, number, and orientation of cluster-bound bases. As an efficient and universal means of exciting AgDNA fluorescence, UV excitation should prove useful in detailed studies of AgDNA photophysics and in identification and purification of the most stable AgDNA fluorophores.

D. Fygenson

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