Biomedical subjects

Neal W Woodbury

Publications and source records attributed to Neal W Woodbury.

17 recordsLinked to original sources

PubMed2006

Signaling aptamers created using fluorescent nucleotide analogues.

A new approach to creating fluorescent signaling aptamers using fluorescent nucleotide analogues is presented. The fluorescence quantum yield of nucleotide analogues such as 2-aminopurine strongly depends on base stacking interactions when incorporated into double or single stranded DNA. This property is used to generate a binding-specific fluorescence signal. Aptamers for human alpha-thrombin, immunoglobulin E, and platelet-derived growth factor B were modified with fluorescent nucleotide analogues in positions that undergo conformational changes. The resulting signaling aptamers show a specific, binding-induced increase in the fluorescence signal of up to 30-fold. Conformation-changing positions in these aptamers were identified by screening a set of modified aptamer sequences that each included a fluorescent nucleotide analogue at a different position. The positions for these modifications were estimated by modeling the aptamer secondary structure. It is likely that this approach to producing fluorescent signaling aptamers is of general use for protein-binding aptamers because of their "induced fit" binding mechanism.

Aptamers, Nucleotide↗

PubMed2006

Mechanism of carotenoid singlet excited state energy transfer in modified bacterial reaction centers.

Ultrafast transient laser spectroscopy has been used to investigate carotenoid singlet excited state energy transfer in various Rhodobacter (Rb.) sphaeroides reaction centers (RCs) modified either genetically or chemically. The pathway and efficiency of energy transfer were examined as a function of the structures and energies of the donor and acceptor molecules. On the donor side, carotenoids with various extents of pi-electron conjugation were examined. RCs studied include those from the anaerobically grown wild-type strain containing the carotenoid spheroidene, which has 10 conjugated carbon-carbon double bonds; the GA strain containing neurosporene, which has nine conjugated double bonds; and aerobically grown wild-type cells, as well as aerobically grown H(M182)L mutant, both containing the carbonyl-containing carotenoid spheroidenone, which has 11 conjugated double bonds. By varying the structure of the carotenoid, we observed the effect of altering the energies of the carotenoid excited states on the rate of energy transfer. Both S(1)- and S(2)-mediated carotenoid-to-bacteriochlorophyll energy transfer processes were observed. The highest transfer efficiency, from both the S(1) and S(2) states, was observed using the carotenoid with the shortest chain. The S(1)-mediated carotenoid-to- bacteriochlorophyll energy transfer efficiencies were determined to be 96%, 84%, and 73% for neurosporene, spheroidene, and spheroidenone, respectively. The S(2)-mediated energy transfer efficiencies follow the same trend but could not be determined quantitatively because of limitations in the time resolution of the instrumentation. The dependence of the energy transfer rate on the energetics of the energy transfer acceptor was verified by performing measurements with RCs from the H(M182)L mutant. In this mutant, the bacteriochlorophyll (denoted B(B)) located between the carotenoid and the RC special pair (P) is replaced by a bacteriopheophytin (denoted phi(B)), where the Q(X) and Q(Y) bands of phi(B) are 1830 and 1290 cm(-1), respectively, higher in energy than those of B(B). These band shifts associated with phi(B) in the H(M182)L mutant significantly alter the spectral overlap between the carotenoid and phi(B), resulting in a significant decrease of the transfer efficiency from the carotenoid S(1) state to phi(B). This leaves energy transfer from the carotenoid S(2) state to phi(B) as the dominant channel. Largely because of this change in mechanism, the overall efficiency of energy transfer from the carotenoid to P decreases to less than 50% in this mutant. Because the spectral signature of phi(B) is different from that of B(A) in this mutant, we were able to demonstrate clearly that the carotenoid-to-P energy transfer is via phi(B). This finding supports the concept that, in wild-type RCs, the carotenoid-to-P energy transfer occurs through the cofactor located at the B(B) position.

Carotenoids↗

PubMed2006

Electronic transitions of the Soret band of reaction centers from Rhodobacter sphaeroides studied by femtosecond transient absorbance spectroscopy.

The Soret band of reaction centers from Rhodobacter sphaeroides has been systematically studied using femtosecond transient absorption spectroscopy. When the excitation wavelength was scanned over the entire Soret band, the approximate absorption spectra of the bacteriochlorophyll dimer, the monomer bacteriochlorophylls, and the bacteriopheophytins within the Soret band were determined by analyzing the ground state bleaching with about 100 fs resolution. The main contribution of H is on the blue end of the spectrum, peaking near 350 nm, P absorbs mostly on the red side of the spectrum, but probably has multiple bands, and the main absorbance of B likely lies between H and P, overlapping with P on the red side (particularly near 390 nm). The energy transfer from B to P in the QY band takes about 300 fs when Soret-band excitation is used and the time constant of overall energy transfer from H to B to P in the QY band when H is specifically excited near 350 nm is about 500 fs. Internal conversion after Soret-band excitation is the rate-limiting step for the energy-transfer process. The time constant of internal conversion for B and P is less than 300 fs, and for H it is about 500 fs.

Electrons↗

PubMed2006

Synthesis and characterization of peptide grafted porous polymer microstructures.

A scanning laser system has been used to generate three-dimensional trimethylolpropane trimethacrylate (TRIM) cross-linked poly(2-hydroxylethyl methacrylate) polymer microstructures through azo-bis(isobutyro)nitrile (AIBN) photopolymerization using a 20 x 0.5 NA microscope objective and 365 nm laser excitation. Macropores are observed to form without the use of porogens in regions of highest light flux. This is attributed to phase separation, which results from differences in monomer reactivity and miscibility. The microstructures were aminated and then protected with the photolabile protective group 6-nitroveratryloxycarbonyl (NVOC). This made it possible to selectively modify the microstructures with the same scanning laser system that was used to fabricate them, resulting in peptide grafted three-dimensional porous microstructures. On the basis of the absorbance of the dibenzofulvene-piperidine, these structures have an amine site density of approximately 0.1 nmol/feature. MALDI-TOF MS was used to characterize peptide photografted microstructures. N-Tris(2,4,6-trimethoxyphenyl)phosphonium (TMPP) labeling of the peptides greatly enhanced detection and allowed post-source decay sequencing of the peptides from the microstructures. The techniques described could be used to generate three-dimensional peptide grafted porous scaffolds for tissue engineering applications.

Benzaldehydes↗

PubMed2005

Environmental control of primary photochemistry in a mutant bacterial reaction center.

The core structure of the photosynthetic reaction center is quasisymmetric with two potential pathways (called A and B) for transmembrane electron transfer. Both the pathway and products of light-induced charge separation depend on local electrostatic interactions and the nature of the excited states generated at early times in reaction centers isolated from Rhodobacter sphaeroides. Here transient absorbance measurements were recorded following specific excitation of the Q(y)() transitions of P (the special pair of bacteriochlorophylls), the monomer bacteriochlorophylls (B(A) and B(B)), or the bacteriopheophytins (H(A) and H(B)) as a function of both buffer pH and detergent in a reaction center mutant with the mutations L168 His to Glu and L170 Asn to Asp in the vicinity of P and B(B). At a low pH in any detergent, or at any pH in a nonionic detergent (Triton X-100), the photochemistry of this mutant is faster than, but similar to, wild type (i.e. electron transfer occurs along the A-side, 390 nm excitation is capable of producing short-lived B-side charge separation (B(B)(+)H(B)(-)) but no long-lived B(B)(+)H(B)(-) is observed). Certain buffering conditions result in the stabilization of the B-side charge separated state B(B)(+)H(B)(-), including high pH in the zwitterionic detergent LDAO, even following excitation with low energy photons (800 or 740 nm). The most striking result is that conditions giving rise to stable B-side charge separation result in a lack of A-side charge separation, even when P is directly excited. The mechanism that links B(B)(+)H(B)(-) stabilization to this change in the photochemistry of P in the mutant is not understood, but clearly these two processes are linked and highly sensitive to the local electrostatic environment produced by buffering conditions (pH and detergent).

Buffers↗

PubMed2005

Development and characterization of green fluorescent protein mutants with altered lifetimes.

Multiple-probe fluorescence imaging applications demand an ever-increasing number of resolvable probes, and the use of fluorophores with resolvable fluorescence lifetimes can help meet this demand. Green fluorescent protein (GFP) and its variants have been widely used in spectrally resolved multiprobe imaging, but as yet, there has not been a systematic set of mutants generated with resolvable lifetimes. Therefore, to generate such mutants, we have utilized error-prone PCR and fluorescence lifetime imaging to screen for mutants of UV-excited green fluorescent protein (GFPuv) that exhibit altered fluorescence decay lifetimes. This has resulted in the isolation of GFPuv mutants displaying at least three distinctly different lifetimes in the range of 1.9-2.8 ns. Mutation of Y145 to either histidine or cysteine was found to shift the fluorescence lifetime of GFPuv from 3.03 +/- 0.03 to 2.78 +/- 0.05 ns for the Y145H mutant and to 2.74 +/- 0.05 ns for Y145C. Some of the shorter-lifetime mutants exhibited excitation peaks that were red-shifted relative to their maximal absorption, indicating that the mutations allowed the adoption of additional conformations relative to wtGFPuv. The utility of these mutants for applications in simultaneous imaging and quantification is shown by the ability to quantify the composition of binary mixtures in time-resolved images using a single detector channel. The application of the screening method for generating lifetime mutants of other fluorescent proteins is also discussed.

Animals↗

PubMed2005

Light-directed movement of polymer microstructures.

Light has been used to induce photochemical changes in the surface chemistry of porous polymer microstructures giving rise to a substantial change in volume. When illumination is asymmetric, this results in light-directed motion of the structure. Swellable trimethylolpropane trimethacrylate cross-linked poly(2-hydroxylethyl methacrylate) microstructures were constructed by azo-bis-isobutyronitrile photopolymerization using a 20 x 0.5 NA microscope objective and 365 nm laser excitation. Structures were aminiated with glycine and protected with the photolabile group 4-nitroveratryloxycarbanyl (NVOC). Addition of NVOC resulted in a volume increase >10% when performed in the solvent N,N'-dimethylformamide. Photochemical cleavage of NVOC using asymmetric illumination of a cone-shaped microstructure with a 365 nm laser induced polymer shrinkage in excess of 4% at the base of the cone and resulted in a maximum velocity of 1 mm/s at the tip of the cone. Symmetric illumination gave rise to displacement of solvent from the microstructure due to shrinkage with a velocity in excess of 0.01 mm/s. This system could in principle be used for light-directed movement of micromechanical systems, optical control of microfluidics, or light activated chemical delivery.

Journal Article↗

PubMed2004

Photolysis and recombination of adenosylcobalamin bound to glutamate mutase.

Ultrafast spectroscopic measurements are used to determine the kinetics of homolysis and recombination for adenosylcobalamin bound in the active site of glutamate mutase. These are the first such measurements on an adenosylcobalamin-dependent enzyme. A short-lived intermediate is formed prior to formation of the cob(II)alamin radical. This intermediate was not observed upon photolysis of adenosylcobalamin in free solution. The intrinsic rate constant for geminate recombination for adenosylcobalamin bound to glutamate mutase is 1.08 +/- 0.10 ns-1, only 16% smaller than the rate constant measured in free solution, 1.39 +/- 0.06 ns-1, suggesting the protein does not greatly perturb the stability of the cobalt-carbon bond upon binding the coenzyme.

Cobamides↗

PubMed2003

Optical processing of bacterial libraries for directed evolution.

Selection of phenotypically distinct bacterial colonies on a Petri dish is typically performed by one of two methods: chemical or mechanical. Chemical methods (e.g., antibiotic selection) rely on inherent growth advantages of the unique phenotypes desired and thus have limited applicability. Mechanical methods are generally slow and require relatively large colonies (typically hundreds of colonies per plate). Here the use of imaged light to select bacterial colonies is explored, employing either photodynamic therapy agents or a ferrochelatase mutation in combination with porphyrin precursors to sensitize the bacteria to light and a computer-controlled light projection system to illuminate some bacterial colonies while leaving others in the dark. A CCD camera was used to distinguish between bacteria expressing green fluorescent protein (GFP) from nonfluorescent colonies. The fluorescence image from the camera was then used to create a virtual masking image for photoselection. Using a simple commercial projector it was possible to confer a 56-fold selective advantage to colonies expressing GFP. This represents a potentially powerful tool in directed evolution experiments using large libraries.

Cell Division↗

PubMed2003

Energy trapping and detrapping in reaction center mutants from Rhodobacter sphaeroides.

Time-resolved fluorescence of chromatophores isolated from strains of Rhodobacter sphaeroides containing light harvesting complex I (LHI) and reaction center (RC) (no light harvesting complex II) was measured at several temperatures between 295 K and 10 K. Measurements were performed to investigate energy trapping from LHI to the RC in RC mutants that have a P/P(+) midpoint potential either above or below wild-type (WT). Six different strains were investigated: WT + LHI, four mutants with altered RC P/P(+) midpoint potentials, and an LHI-only strain. In the mutants with the highest P/P(+) midpoint potentials, the electron transfer rate decreases significantly, and at low temperatures it is possible to directly observe energy transfer from LHI to the RC by detecting the fluorescence kinetics from both complexes. In all mutants, fluorescence kinetics are multiexponential. To explain this, RC + LHI fluorescence kinetics were analyzed using target analysis in which specific kinetic models were compared. The kinetics at all temperatures can be well described with a model which accounts for the energy transfer between LHI and the RC and also includes the relaxation of the charge separated state P(+)H(A)(-), created in the RC as a result of the primary charge separation.

Cell Membrane↗

PubMed2003

Excitonic interactions in wild-type and mutant PSI reaction centers.

Femtosecond excitation of the red edge of the chlorophyll a Q(Y) transition band in photosystem I (PSI), with light of wavelength > or = 700 nm, leads to wide transient (subpicosecond) absorbance changes: positive DeltaA between 635 and 665 nm, and four negative DeltaA bands at 667, 675, 683, and 695 nm. Here we compare the transient absorbance changes after excitation at 700, 705, and 710 nm at 20 K in several PSI preparations of Chlamydomonas reinhardtii where amino acid ligands of the primary donor, primary acceptor, or connecting chlorophylls have been mutated. Most of these mutations influence the spectrum of the absorbance changes. This supports the view that the chlorophylls of the electron transfer chain as well as the connecting chlorophylls are engaged in the observed absorbance changes. The wide absorption spectrum of the electron transfer chain revealed by the transient measurements may contribute to the high efficiency of energy trapping in photosystem 1. Exciton calculations, based on the recent PSI structure, allow an assignment of the DeltaA bands to particular chlorophylls: the bands at 675 and 695 nm to the dimers of primary acceptor and accessory chlorophyll and the band at 683 nm to the connecting chlorophylls. The subpicosecond transient absorption bands decay may reflect rapid charge separation in the PSI reaction center.

Amino Acid Substitution↗

PubMed2003

Characterization of Chlorobium tepidum chlorosomes: a calculation of bacteriochlorophyll c per chlorosome and oligomer modeling.

The bacteriochlorophyll (Bchl) c content and organization was determined for Chlorobium (Cb.) tepidum chlorosomes, the light-harvesting complexes from green photosynthetic bacteria, using fluorescence correlation spectroscopy and atomic force microscopy. Single-chlorosome fluorescence data was analyzed in terms of the correlation of the fluorescence intensity with time. Using this technique, known as fluorescence correlation spectroscopy, chlorosomes were shown to have a hydrodynamic radius (Rh) of 25 +/- 3.2 nm. This technique was also used to determine the concentration of chlorosomes in a sample, and pigment extraction and quantitation was used to determine the molar concentration of Bchl c present. From these data, a number of approximately 215,000 +/- 80,000 Bchl c per chlorosome was determined. Homogeneity of the sample was further characterized by dynamic light scattering, giving a single population of particles with a hydrodynamic radius of 26.8 +/- 3.7 nm in the sample. Tapping-mode atomic force microscopy (TMAFM) was used to determine the x,y,z dimensions of chlorosomes present in the sample. The results of the TMAFM studies indicated that the average chlorosome dimensions for Cb. tepidum was 174 +/- 8.3 x 91.4 +/- 7.7 x 10.9 +/- 2.71 nm and an overall average volume 90,800 nm(3) for the chlorosomes was determined. The data collected from these experiments as well as a model for Bchl c aggregate dimensions was used to determine possible arrangements of Bchl c oligomers in the chlorosomes. The results obtained in this study have significant implications on chlorosome structure and architecture, and will allow a more thorough investigation of the energetics of photosynthetic light harvesting in green bacteria.

Bacterial Proteins↗

PubMed2003

Single molecule detection of DNA looping by NgoMIV restriction endonuclease.

Single molecule fluorescence resonance energy transfer (FRET) and fluorescence correlation spectroscopy were used to investigate DNA looping by NgoMIV restriction endonuclease. Using a linear double-stranded DNA (dsDNA) molecule labeled with a fluorescence donor molecule, Cy3, and fluorescence acceptor molecule, Cy5, and by varying the concentration of NgoMIV endonuclease from 0 to 3 x 10(-6) M, it was possible to detect and determine diffusion properties of looped DNA/protein complexes. FRET efficiency distributions revealed a subpopulation of complexes with an energy transfer efficiency of 30%, which appeared upon addition of enzyme in the picomolar to nanomolar concentration range (using 10(-11) M dsDNA). The concentration dependence, fluorescence burst size analysis, and fluorescence correlation analysis were all consistent with this subpopulation arising from a sequence specific interaction between an individual enzyme and a DNA molecule. A 30% FRET efficiency corresponds to a distance of approximately 65 A, which correlates well with the distance between the ends of the dsDNA molecule when bound to NgoMIV according to the crystal structure of this complex. Formation of the looped complexes was also evident in measurements of the diffusion times of freely diffusing DNA molecules with and without NgoMIV. At very high protein concentrations compared to the DNA concentration, FRET and fluorescence correlation spectroscopy results revealed the formation of larger DNA/protein complexes.

Binding Sites↗

PubMed2003

Single-molecule fluorescence spectroscopy of TOTO on poly-AT and poly-GC DNA.

Excited state lifetime and amplitude measurements were made on thiazole orange dimer (TOTO), a dimeric DNA-intercalating fluorophore, at single-molecule concentrations. As expected from previous study, the excited state lifetime of TOTO intercalated in DNA is dependent on the sequence of the double-stranded DNA, having values of 2.2 ns in poly-GC DNA and 1.8 ns in poly-AT DNA. The distribution of excited state lifetimes of single molecules of TOTO intercalated into oligonucleotides having varying proportions of poly-GC sequences relative to poly-AT sequences were analyzed as a function of the fraction of poly-GC. By using excited state lifetime distributions from the purely GC and purely AT oligonucleotides as a basis set, it was possible to estimate the GC content of oligonucleotides with intermediate GC composition to within a few percent error. This serves as a model for the analysis of equilibrium binding distributions in DNA using single-molecule methods.

Base Sequence↗

PubMed2003

TOTO binding affinity analysis using single-molecule fluorescence spectroscopy.

The sequence dependence of the double-stranded DNA (dsDNA)-binding affinity of TOTO, a thiazole orange dimer that functions as a DNA-intercalating fluorophore, was measured using single-molecule methods. An analysis was performed of the distribution of excited-state lifetimes of single molecules of TOTO intercalated into dsDNA fragments containing four-base pair sequences shown previously to have high affinity for TOTO under conditions used in nuclear magnetic resonance (NMR) spectroscopy. For the current studies, the putative binding sites were located centrally in 30-base pair-long dsDNA fragments in which the remaining sequence was either all poly-AT or poly-GC. The lifetime of TOTO fluorescence when bound to these fragments was entirely determined by the background sequence, i.e. DNA fragments with a poly-AT background predominantly gave a fluorescence lifetime of 1.7 ns, whereas DNA fragments with a poly-GC background gave a lifetime of 2.0 ns, independent of the presence or absence of the putative binding sequence. By performing competitive binding experiments in which these DNA fragments competed for TOTO binding with pure poly-AT fragments and using single-molecule fluorescence methods to determine the number of each type of DNA fragment having a TOTO bound in an equilibrium mixture, the relative binding affinity of each putative binding site was determined. The results of these experiments showed clearly that TOTO has no preference for binding to the putative binding sites over binding poly-AT or poly-GC under the conditions of these measurements. This suggests that there is very little sequence dependence of TOTO binding under conditions that would likely predominate in most biological applications of this intercalating dye.

Base Sequence↗

PubMed2002

DNA-binding interactions and conformational fluctuations of Tc3 transposase DNA binding domain examined with single molecule fluorescence spectroscopy.

The fluorescent dye tetramethylrhodamine (TMR) was conjugated to a synthetic peptide containing the sequence-specific DNA binding domain of Tc3 transposase. Steady-state and single molecule fluorescence spectroscopy was used to investigate protein conformational fluctuations and the thermodynamics of binding interactions. Evidence is presented to show that the TMR-Tc3 conjugate exists in at least two conformational states. The most stable conformation is one in which the TMR fluorescence is quenched. Upon binding to DNA, the total fluorescence from TMR-Tc3 increases by three- to fourfold. Single molecule measurements of TMR-Tc3 bound to DNA shows that this complex also fluctuates between a fluorescent and quenched form. The fluorescent form of the conjugate is stabilized when bound to DNA, and this accounts for part of the increase in total fluorescence. In addition, the inherent photodynamics of the dye itself is also altered (e.g., fluorescent lifetime or triplet yield) in such a way that the total fluorescence from the conjugate bound to DNA is enhanced relative to the unbound form.

Animals↗

PubMedDate not supplied

Multiphoton excitation of fluorescent DNA base analogs.

Multiphoton excitation was used to investigate properties of the fluorescent DNA base analogs, 2-aminopurine (2AP) and 6-methylisoxanthopterin (6MI). 2-aminopurine, a fluorescent analog of adenine, was excited by three-photon absorption. Fluorescence correlation measurements were attempted to evaluate the feasibility of using three-photon excitation of 2AP for DNA-protein interaction studies. However, high excitation power and long integration times needed to acquire high signal-to-noise fluorescence correlation curves render three-photon excitation FCS of 2AP not very useful for studying DNA base dynamics. The fluorescence properties of 6-methylisoxanthopterin, a guanine analog, were investigated using two-photon excitation. The two-photon absorption cross-section of 6MI was estimated to be about 2.5 x 10(-50) cm(4)s (2.5 GM units) at 700 nm. The two-photon excitation spectrum was measured in the spectral region from 700 to 780 nm; in this region the shape of the two-photon excitation spectrum is very similar to the shape of single-photon excitation spectrum in the near-UV spectral region. Two-photon excitation of 6MI is suitable for fluorescence correlation measurements. Such measurements can be used to study DNA base dynamics and DNA-protein interactions over a broad range of time scales.

2-Aminopurine↗