William S. Wessels

Human GLTP: Three distinct functions for the three tryptophans in a novel peripheral amphitropic fold

Human glycolipid transfer protein (GLTP) serves as the GLTP-fold prototype, a novel, to our knowledge, peripheral amphitropic fold and structurally unique lipid binding motif that defines the GLTP superfamily. Despite conservation of all three intrinsic Trps in vertebrate GLTPs, the Trp functional role(s) remains unclear. Herein, the issue is addressed using circular dichroism and fluorescence spectroscopy along with an atypical Trp point mutation strategy. Far-ultraviolet and near-ultraviolet circular dichroism spectroscopic analyses showed that W96F-W142Y-GLTP and W96Y-GLTP retain their native conformation and stability, whereas W85Y-W96F-GLTP is slightly altered, in agreement with relative glycolipid transfer activities of >90%, ∼85%, and ∼45%, respectively. In silico three-dimensional modeling and acrylamide quenching of Trp fluorescence supported a nativelike folding conformation. With the Trp⁹⁶-less mutants, changes in emission intensity, wavelength maximum, lifetime, and time-resolved anisotropy decay induced by phosphoglyceride membranes lacking or containing glycolipid and by excitation at different wavelengths along the absorption-spectrum red edge indicated differing functions for W142 and W85. The data suggest that W142 acts as a shallow-penetration anchor during docking with membrane interfaces, whereas the buried W85 indole helps maintain proper folding and possibly regulates membrane-induced transitioning to a glycolipid-acquiring conformation. The findings illustrate remarkable versatility for Trp, providing three distinct intramolecular functions in the novel amphitropic GLTP fold.

Electrophoretic mobility of weakly-charged (dipolar) hydrogels in water: contribution of hydrogen-bonding in the solvent dipole layer.

Counterintuitive observations by dynamic light-scattering experiments of negative electrophoretic mobility in uncharged, lightly charged, and later, densely-charged hydrogel nanoparticles are presented. A tentative theory, emphasizing the roles of electric field energy density and induced dipole moments in the dipolar and hydrogen-bonding solvent layer surrounding the particle, is introduced to explain and rationalize these observations. Addition of co-solvent glycine seems to produce a Kohlrausch boundary regulating effect which again illustrates the importance of the dipole layer and hydrogen bonds within it. Further alternative theories involving electric field gradients are discussed which may be relevant to other uncharged systems (such as gold nanoparticles). A contribution to the dipolar solvent-induced mobility is derived in Appendix A. A proposal for a new treatment of traditional (i.e. charged colloid particle) electrokinetic phenomena is given in a second Appendix (Appendix B).

Photophysics of EGFP (E222H) Mutant, with Comparisons to Model Chromophores: Excited State pK’s, Progressions, Quenching and Exciton Interaction

AbstractA novel version of the well-known and commercially successful Green Fluorescent Protein (GFP) variant known as EGFP, with an introduced E222H mutation, was produced in this laboratory. Given the current state of hypotheses about the role of glutamate 222, and the observed dominance of the phenolate absorption with an E222H variant observed from earlier study, the new mutant was considered a natural choice to investigate more fully the acid-base behavior of the chromophore in absorption and fluorescence. The bulk of this investigation concerns fitting the excitation, emission and absorption spectra to vibrational progressions of a novel ‘q-deformed’ type at various values of pH, and protein concentration. From these data, and from temperature-dependent fluorescence lifetime data and other experiments (with lanthanide doped gels into which H/EGFP is embedded), we construct a picture of excited inter- state conversion mechanisms, and quenching mechanisms, that attempts to explain many features of the GFP system. Graphical AbstractHypothetical proton current loop (orange) upon excitation; electron motion in purple H/EGFP. Solid boxes about waters project toward viewer, dashed boxes project away