William H Sawyer

Publications (7)20.78 Total impact

• Article: The thiourea group modulates the fluorescence emission decay of fluorescein-labeled molecules.

  Nectarios Klonis, William H Sawyer
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  ABSTRACT: We have investigated the spectral properties and emission characteristics of fluorescein-5-thiocarbamoyl-N,N'-caproate (FITC-ACA) to examine the origin of the complex emission decay often observed in fluorescein-labeled molecules. The covalent attachment of fluorescein to epsilon-amino-n-caproic acid does not perturb the prototropic transitions of the chromophore or the general fluorescence characteristics of the various prototropic forms. However, both the monoanion and dianion forms of FITC-ACA are quenched relative to free fluorescein and exhibit a complex emission decay that is described by two discrete lifetimes. The thiourea group that links the chromophore to the caproic acid is shown to modulate the emission properties of the FITC-ACA. We show that the emission decay can also be analyzed using the asymmetric distribution model of Alcala et al. In this analysis, the tauL and tauu parameters that represent the lower and upper lifetime limits of the distribution reflect the quenched (0 ns) and unquenched lifetimes, respectively. The beta parameter that describes the distribution of lifetimes between the two limiting states can be related to the quenching efficiency of the thiourea group and to the structure and dynamics of the FITC-ACA molecule.
  Photochemistry and Photobiology 06/2003; 77(5):502-9. · 2.41 Impact Factor
• Article: Unfolding of class A amphipathic peptides on a lipid surface.

  Andrew H A Clayton, Andra G Vultureanu, William H Sawyer
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  ABSTRACT: The folding of polypeptides associated with biomembranes is a ubiquitous phenomenon, yet the thermodynamics underlying the process are poorly understood. In the present work we examine the unfolding of a series of alpha-helical amphipathic membrane-associated peptides using guanidine hydrochloride as a denaturant. The peptides are based on the class A amphipathic helix motif, and each contains a single tryptophan at sequence position 2, 3, 7, 12, or 14. The isothermal unfolding process was monitored by circular dichroism ellipticity at 222 nm to monitor changes in the helical structure of the peptide. Tryptophan fluorescence was used to probe the local changes in the environment about the indole fluorophore. The unfolding curves generated from the two experimental techniques for each peptide-lipid complex were non-coincidental, suggesting the presence of stable intermediate(s) in the unfolding. A three-state model could adequately account for the data and yielded parameters which were consistent with the presence of a partially folded intermediate structure which (i) is closer in Gibb's free energy to the folded state than the unfolded state and (ii) retains much of the interfacial and amphipathic character of the folded state. Denaturant-induced peptide dissociation from the peptide-lipid complexes was found to be negligible as confirmed by size exclusion chromatography. The results are compared with related thermodynamic data and discussed in terms of current models of peptide folding at membrane interfaces.
  Biochemistry 03/2003; 42(6):1747-53. · 3.42 Impact Factor
• Article: The central domain of Escherichia coli TyrR is responsible for hexamerization associated with tyrosine-mediated repression of gene expression.

  Mathew P Dixon, Richard N Pau, Geoffrey J Howlett, David E Dunstan, William H Sawyer, Barrie E Davidson
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  ABSTRACT: TyrR from Escherichia coli regulates the expression of genes for aromatic amino acid uptake and biosynthesis. Its central ATP-hydrolyzing domain is similar to conserved domains of bacterial regulatory proteins that interact with RNA polymerase holoenzyme associated with the alternative sigma factor, sigma(54). It is also related to the common module of the AAA+ superfamily of proteins that is involved in a wide range of cellular activities. We expressed and purified two TyrR central domain polypeptides. The fragment comprising residues 188-467, called TyrR-(188-467), was soluble and stable, in contrast to that corresponding to the conserved core from residues 193 to 433. TyrR-(188-467) bound ATP and rhodamine-ATP with association constants 2- to 5-fold lower than TyrR and hydrolyzed ATP at five times the rate of TyrR. In contrast to TyrR, which is predominantly dimeric at protein concentrations less than 10 microm in the absence of ligands, or in the presence of ATP or tyrosine alone, TyrR-(188-467) is a monomer, even at high protein concentrations. Tyrosine in the presence of ATP or ATPgammaS promotes the oligomerization of TyrR-(188-467) to a hexamer. Tyrosine-dependent repression of gene transcription by TyrR therefore depends on ligand binding and hexamerization determinants located in the central domain polypeptide TyrR-(188-467).
  Journal of Biological Chemistry 07/2002; 277(26):23186-92. · 4.77 Impact Factor
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  Article: Site-specific tryptophan fluorescence spectroscopy as a probe of membrane peptide structure and dynamics.

  Andrew H A Clayton, William H Sawyer
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  ABSTRACT: The fluorescence from tryptophan contains valuable information about the environment local to the indole side-chain. This environment sensitivity coupled with the ability to synthetically or genetically incorporate a single tryptophan residue at specific sites in a polypeptide sequence has provided the membrane biophysicist with powerful tools for examining the structure and dynamics of membrane peptides and proteins. Here we briefly review the use of site-specific tryptophan fluorescence spectroscopy to probe aspects of peptide orientation, structure, and dynamics in lipid bilayers, focusing on recent developments in the literature.
  European Biophysics Journal 04/2002; 31(1):9-13. · 2.14 Impact Factor
• Article: Spectral Properties of Fluorescein in Solvent‐Water Mixtures: Applications as a Probe of Hydrogen Bonding Environments in Biological Systems

  Nectarios Klonis, Andrew H. A. Clayton, Edward W. Voss Jr, William H. Sawyer
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  ABSTRACT: Although fluorescein is a widely used fluorescent probe in the biosciences, the effect of solvent environment on its spectral properties is poorly understood. In this paper we explore the use of fluorescein as a probe of the state of hydrogen bonding in its local environment. This application is based on the observation, originally made by Martin (Chem. Phys. Lett. 35, 105–111, 1975), that the absorption maximum of fluorescein undergoes substantial shifts in organic solvents related to the hydrogen bonding power of the solvents. We have extended this work by studying the spectral properties of the dianion form of the probe in solvent–water mixtures. We show that the magnitude of the shift correlates with the α and β parameters of Kamlet and Taft (J. Am. Chem. Soc. 98, 377–383; 2886–2894, 1976), which provide a scale of the hydrogen bond donor acidities and acceptor basicities, respectively, of the solvents. In solvent–water mixtures, these shifts reflect general effects of the solvents on the hydrogen bonding environment of the fluorescein through water–solvent hydrogen bonding and specific effects due to fluorescein–solvent hydrogen bonding. Indeed, both the absorption and fluorescence properties appear to be dominated by these effects indicating that the spectral shifts of the dianion can be used as an indicator of its hydrogen bonding environment. We discuss the application of fluorescein as a probe of hydrogen bonding in the microenvironment immediately surrounding the fluorophore, and we illustrate the effect with reference to the fluorescein–antifluorescein antibody complex where it appears that antibodies selected during the immune response possess binding sites that are increasingly dehydrated and hydrophobic.
  Photochemistry and Photobiology 04/1998; 67(5):500 - 510. · 2.41 Impact Factor
• Article: Spectral properties of the prototropic forms of fluorescein in aqueous solution

  Nectarios Klonis, William H. Sawyer
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  ABSTRACT: The commonly used fluorescent probe, fluorescein, can exist in seven prototropic forms. We have used global analysis procedures to reanalyze the absorption data of Diehl and Horchak-Morris (Talanta 34, 739–741, 1987) in terms of five alternative ionization models. We identify the forms of fluorescein present in aqueous solution and the pK a of each ionisation transition. The pKa values of the neutral xanthene, carboxylic acid, and cationic xanthene groups are 6.3, 3.1–3.4, and 3.1–3.4, respectively, and the pKa value of lactonization is 2.4. As a consequence, the neutral form of fluorescein is a mixture of the lactone (70%), zwitterionic (15%), and quinoid (15%) forms. A knowledge of the forms present in solution permits the characterization of their spectral properties. It is shown that the quinoid and monoanion forms have similar absorption spectra, while the zwitterion spectrum is similar to that of the cation but blue-shifted by 3 nm. The emission spectra of the monoanion and quinoid forms are also identified and shown to be similar but not identical. A model for the excited-state reactions of fluorescein is presented.
  Journal of Fluorescence 08/1996; 6(3):147-157. · 2.11 Impact Factor
• Article: Federation of Asian and Oceanian Biochemists and Molecular Biologists Incorporated (FAOBMB Inc.): a brief history.

  Jisnuson Svasti, William H Sawyer
  International Union of Biochemistry and Molecular Biology Life 58(5-6):280-2. · 3.51 Impact Factor