Biomedical subjects

Jay R Unruh

Publications and source records attributed to Jay R Unruh.

10 recordsLinked to original sources

PubMed2006

Conformational flexibility, hydration and state parameter fluctuations of fibroblast growth factor-10: effects of ligand binding.

Differential effects of ligand binding on local and global fibroblast growth factor-10 (FGF-10) flexibility and stability have been investigated utilizing a variety of experimental and computational techniques. Normal mode analysis was used to predict the low frequency motions and regional flexibility of FGF-10. Similarly, regional variations in local folding/unfolding equilibria were characterized with the COREX/BEST algorithm. Experimental adiabatic and isothermal compressibilities of FGF-10 alone and in the presence of polyanions are compared. Furthermore, the effect of polyanions on the coefficient of thermal expansion is compared. Measurements of density, heat capacity, compressibility, and expansibility were combined to calculate experimentally determined volume and enthalpy fluctuations. Global effects of polyanions on FGF-10 flexibility, thermodynamic fluctuations, and hydration vary depending on the size and charge density of the polyanion. Local effects of polyanions were investigated utilizing time-resolved fluorescence spectroscopy and red edge excitation spectroscopy (REES). Increased rigidity of the protein matrix or an increased solvent response surrounding the Trp residues is observed in the presence of polyanions. Similarly, time-resolved spectroscopy reveals increased ground state heterogeneity and increased dipole relaxation on the time scale of fluorescence for FGF-10 in the presence of polyanions. These polyanions increase heterogeneity, global flexibility, and fluctuations while increasing the melting temperature (Tm) of FGF-10.

Crystallography, X-Ray↗

PubMed2006

Effects of ligand binding and oxidation on hinge-bending motions in S-adenosyl-L-homocysteine hydrolase.

Domain motions of S-adenosyl-l-homocysteine (AdoHcy) hydrolase have been detected by time-resolved fluorescence anisotropy measurements. Time constants for reorientational motions in the native enzyme were compared with those for enzymes where key residues were altered by site-directed mutation. Mutations M351P, H353A, and P354A were selected in a hinge region for motion between the open and closed forms of the enzyme, as identified in a previous normal-mode study [Wang et al. (2005) Domain motions and the open-to-closed conformational transition of an enzyme: A normal-mode analysis of S-adenosyl-l-homocysteine hydrolase, Biochemistry 44, 7228-7239]. In wild-type, substrate-free AdoHcy hydrolase (NAD(+) cofactor in each subunit), reorientational motions were detected on time scales of 10-20 and 80-90 ns. The faster motion is attributed to the domain motion, and the slower motion is attributed to the tumbling of the enzyme. The domain motion was also detected for the enzyme complexes E(NADH/3'-keto-adenosine) and E(NAD(+)/3'-deoxyadenosine) but was absent for the complex E(NADH/3'-keto-neplanocin A). The results indicate that AdoHcy hydrolase exists in equilibrium of open and closed structures, with the equilibrium shifted toward the more mobile open form for the substrate-free enzyme, E(NAD(+)), and for intermediates formed early in the catalytic cycle after substrate binding or formed late prior to product release, E(NAD(+)/ligand). However, the strong inhibitor neplanocin A upon binding undergoes oxidation, forming the complex E(NADH/3'-keto-neplanocin). For this complex, which is analogous to the enzyme complex with the central catalytic intermediate, the equilibrium was shifted toward the more rigid closed form. A similar pattern was observed for M351P and P354A mutants. In contrast, the domain motion could not be detected, either in the absence or presence of ligands or with the cofactor in either the oxidized or reduced state, for the H353A protein, suggesting that this mutation changes the hinge-bending dynamics of the enzyme.

Adenosylhomocysteinase↗

PubMed2006

Evaluation of a femtosecond fiber laser for two-photon fluorescence correlation spectroscopy.

This work evaluates a femtosecond fiber laser for use in two-photon fluorescence fluctuation spectroscopy. Fiber lasers present an attractive alternative to Ti:Sapphire systems because of their compact size and portability. Autocorrelation of the second harmonic generation signal from the laser demonstrates that its stability is sufficient for two-photon fluorescence correlation spectroscopy. Fluorescence correlation spectroscopy autocorrelation traces were well fit by a Gaussian-Lorentzian squared model with a beam waist near the diffraction limit for the 810 nm wavelength. A photon counting histogram collected with this system also fit nicely to a single-species model, further demonstrating the quality of the focal shape. The authors conclude that the output from the femtosecond fiber laser is sufficiently stable and has a high enough quality beam shape for fluctuation fluorescence methods, and thus represents an effective, compact, readily portable two-photon excitation source.

Journal Article↗

PubMed2005

Mechanism of interaction of PITPalpha with membranes: conformational changes in the C-terminus associated with membrane binding.

Eukaryotic phosphatidylinositol transfer proteins (PITPs) are composed predominantly of small ( approximately 32 kDa) soluble proteins that bind and transfer a single phospholipid, normally phosphatidylinositol or phosphatidycholine. Two forms, PITPalpha and PITPbeta, which share approximately 80% amino acid sequence similarity, are known. Rat PITPalpha was labeled at specific single reactive Cys residues with I-AEDANS and used to examine PITP-membrane interactions. Upon binding to phospholipid vesicles, PITP labeled with AEDANS at the C-terminus, a region postulated to be involved in membrane binding, shows significant decreases in both steady-state and dynamic fluorescence anisotropy. In contrast, PITPs labeled with AEDANS at sites located distal to the C-terminus show increases in both steady-state and dynamic anisotropy. These results suggest that interaction of PITP with membrane surfaces leads to significant alterations in conformation and perhaps melting of the C-terminal helix.

Binding Sites↗

PubMed2005

Sampling unfolding intermediates in calmodulin by single-molecule spectroscopy.

We used single-pair fluorescence resonance energy transfer (spFRET) measurements to characterize denatured and partially denatured states of the multidomain calcium signaling protein calmodulin (CaM) in both its apo and Ca(2+)-bound forms. The results demonstrate the existence of an unfolding intermediate. A CaM mutant (CaM-T34C-T110C) was doubly labeled with fluorescent probes AlexaFlour 488 and Texas Red at opposing globular domains. Single-molecule distributions of the distance between fluorophores were obtained by spFRET at varying levels of the denaturant urea. Multiple conformational states of CaM were observed, and the amplitude of each conformation was dependent on urea concentration, with the amplitude of an extended conformation increasing upon denaturation. The distributions at intermediate urea concentrations could not be adequately described as a combination of native and denatured conformations, showing that CaM does not denature via a two-state process and demonstrating that at least one intermediate is present. The intermediate conformations formed upon addition of urea were different for Ca(2+)-CaM and apoCaM. An increase in the amplitude of a compact conformation in CaM was observed for apoCaM but not for Ca(2+)-CAM upon the addition of urea. The changes in the single-molecule distributions of CaM upon denaturation can be described by either a range of intermediate structures or by the presence of a single unfolding intermediate that grows in amplitude upon denaturation. A model for stepwise unfolding of CaM is suggested in which the domains of CaM unfold sequentially.

Calcium↗

PubMed2005

DNA aptamer-based bioanalysis of IgE by fluorescence anisotropy.

A rapid, homogeneous aptamer-based bioanalysis is reported for the sensitive detection of immunoglobulin E (IgE) using fluorescence polarization (FP). 5'-End-labeled D17.4 DNA aptamer was used for IgE detection based on the anisotropy differences of the labeled ligand. Two different fluorophores, fluorescein and Texas Red, were used to analyze IgE in the low-nanomolar range with high specificity. Measurable anisotropy changes were observed with a short equilibration time. Analysis of the binding data reveals a possible cooperative binding process in solution. The nature of the fluorophore clearly influences the sensitivity of the analysis more than the tether length used for the dye conjugation. The local fluorophore motion is seen to influence the sensitivity of the FP probe significantly. Texas Red is seen to be relatively more sensitive for this approach and has apparently favorable dye-DNA interactions, and a limit of detection of 350 pM was obtained. Significant temperature dependence of the FP responses has been observed in this work. Ionic composition of the binding buffer also influences the assay sensitivity. The results confirm the promise and potential of similar homogeneous assays for aptamer-based bioanalysis.

Aptamers, Nucleotide↗

PubMed2005

Conformational substates of calmodulin revealed by single-pair fluorescence resonance energy transfer: influence of solution conditions and oxidative modification.

A calmodulin (CaM) mutant (T34,110C-CaM) doubly labeled with fluorescence probes AlexaFluor 488 and Texas Red in opposing domains (CaM-DA) has been used to examine conformational heterogeneity in CaM by single-pair fluorescence resonance energy transfer (spFRET). Burst-integrated FRET efficiencies of freely diffusing CaM-DA single molecules yielded distributions of distance between domains of CaM-DA. We recently reported distinct conformational substates of Ca(2+)-CaM-DA and apoCaM-DA, with peaks in the distance distributions centered at approximately 28 A, 34-38 A, and 55 A [Slaughter et al. (2004) J. Phys. Chem. B 108, 10388-10397]. In the present study, shifts in the amplitudes and center distances of the conformational substates were detected with variation in solution conditions. The amplitude of an extended conformation was observed to change as a function of Ca(2+) over a free Ca(2+) range that is consistent with binding to the high affinity, C-terminal Ca(2+) binding sites, suggesting the existence of communication between lobes of CaM. Lowering pH shifted the relative amplitudes of the conformations, with a marked increase in the presence of the compact conformations and an almost complete absence of the extended conformation. In addition, the single-molecule distance distribution of apoCaM-DA at reduced ionic strength was shifted to longer distance and showed evidence of an increase in conformational heterogeneity relative to apoCaM-DA at physiological ionic strength. Oxidation of methionine residues in CaM-DA produced a substantial increase in the amplitude of the extended conformation relative to the more compact conformation. The results are considered in light of a hypothesis that suggests that electrostatic interactions between charged amino acid side chains play an important role in determining the most stable CaM conformation under varying solution conditions.

Amino Acid Substitution↗

PubMed2005

Orientational dynamics and dye-DNA interactions in a dye-labeled DNA aptamer.

We report the picosecond and nanosecond timescale rotational dynamics of a dye-labeled DNA oligonucleotide or "aptamer" designed to bind specifically to immunoglobulin E. Rotational dynamics in combination with fluorescence lifetime measurements provide information about dye-DNA interactions. Comparison of Texas Red (TR), fluorescein, and tetramethylrhodamine (TAMRA)-labeled aptamers reveals surprising differences with significant implications for biophysical studies employing such conjugates. Time-resolved anisotropy studies demonstrate that the TR- and TAMRA-aptamer anisotropy decays are dominated by the overall rotation of the aptamer, whereas the fluorescein-aptamer anisotropy decay displays a subnanosecond rotational correlation time much shorter than that expected for the overall rotation of the aptamer. Docking and molecular dynamics simulations suggest that the low mobility of TR is a result of binding in the groove of the DNA helix. Additionally, associated anisotropy analysis of the TAMRA-aptamer reveals both quenched and unquenched states that experience significant coupling to the DNA motion. Therefore, quenching of TAMRA by guanosine must depend on the configuration of the dye bound to the DNA. The strong coupling of TR to the rotational dynamics of the DNA aptamer, together with the absence of quenching of its fluorescence by DNA, makes it a good probe of DNA orientational dynamics. The understanding of the nature of dye-DNA interactions provides the basis for the development of bioconjugates optimized for specific biophysical measurements and is important for the sensitivity of anisotropy-based DNA-protein interaction studies employing such conjugates.

Anisotropy↗

PubMed2003

Tyrosine and peptide reorientational mobility in polymer solutions: Time-dependent fluorescence anisotropy measurements.

The ability of peptides to form biologically active conformations that bind to receptors is governed by their dynamics and their propensity to form stable structures. Such factors are consequently important in the design of peptide drugs. Moreover, the stability of such peptides depends on interactions of the peptide with the surrounding matrix. In this article, we study the effect of the polymer poly(vinyl pyrrolidone) (PVP) on the mobility and orientational dynamics of tyrosine and a model peptide, Val-Tyr-Pro-Asn-Gly-Ala (VYPNGA) in glycerol-water solutions. Orientational dynamics are studied experimentally by time-resolved fluorescence anisotropy decays of tyrosine. The presence of PVP leads to the possibility of a distribution of environments for the peptide. The orientational dynamics of tyrosine show that the probe molecule experiences two very different environments. In one, tyrosine rotational motion is weakly coupled to PVP, while in the other, tyrosine interacts strongly with PVP leading to much slower rotational times. The dynamics of VYPNGA are more complex. Fast intramolecular, localized reorientations of the tyrosine are detected. The temperature dependence of the reorientational dynamics of the tyrosine side chain reveal that these motions are shielded from solvent friction. In contrast, global motions of the peptide are severely restricted by PVP, suggesting the ability of the polymer to restrict peptide mobility.

Fluorescence Polarization↗

PubMedDate not supplied

Fluorescence properties of fluorescein, tetramethylrhodamine and Texas Red linked to a DNA aptamer.

We report the picosecond time-scale fluorescence dynamics of a dye-labeled DNA oligonucleotide or "aptamer" designed to bind specifically to Immunoglobulin E. Comparison of the photophysics of Texas Red (TR), fluorescein and 5'-carboxytetramethylrhodamine (TAMRA)-labeled aptamers reveals surprising differences with significant implications for measurements of oligonucleotide structure and dynamics. The fluorescence decay of the TR-aptamer is a simple single exponential with a weak temperature dependence. The fluorescence decay of the fluorescein-aptamer (fl-aptamer) is pH dependent and displays a complex temperature dependence with significant changes on melting of the aptamer tertiary structure. Despite its similarities to TR, TAMRA is strongly quenched when conjugated to the aptamer and displays complex fluorescence kinetics best described by a distributed rate model. Using the maximum entropy method, we have discovered two highly temperature-dependent fluorescence lifetimes for the TAMRA-aptamer. One of these lifetimes is similar to that of free TAMRA and displays the same temperature dependence. The other lifetime is quenched and displays a temperature dependence characteristic of a charge transfer reaction. These data set TR apart as an attractive alternative to TAMRA and fluorescein for studies such as fluorescence polarization and fluorescence resonance energy transfer, where environmental sensitivity of the probe is not desired.

DNA↗