Single tryptophan mutants of FtsZ: nucleotide binding/exchange and conformational transitions.

Cell division protein FtsZ cooperatively self-assembles into straight filaments when bound to GTP. A set of conformational changes that are linked to FtsZ GTPase activity are involved in the transition from straight to curved filaments that eventually disassemble. In this work, we characterized the fluorescence of single Trp mutants as a reporter of the predicted conformational changes between the GDP- and GTP-states of Escherichia coli FtsZ.

Optical studies of single-tryptophan B. licheniformis beta-lactamase variants.

beta-lactamases (penicillinases) are important complicating factors in bacterial infections and excellent theoretical and experimental models in protein structure, dynamics and evolution. Bacillus licheniformis exo-small penicillinase (ESP) is a Class A beta-lactamase with three tryptophan residues, one located in each of the two protein domains and one located in the interface between domains. To determine the tryptophan contribution to the ESP UV-absorption, circular dichroism, and steady-state and time-resolved fluorescence, four Trp-->Phe mutants were prepared and characterized.

4',6-Diamidino-2-phenylindole (DAPI) induces bundling of Escherichia coli FtsZ polymers inhibiting the GTPase activity.

FtsZ (Filamentous temperature sensitivity Z) cell division protein from Escherichia coli binds the fluorescence probe DAPI. Bundling of FtsZ was facilitated in the presence of DAPI, and the polymers in solution remained polymerized longer time than the protofilaments formed in the absence of DAPI. DAPI decreased both the maximal velocity of the GTPase activity and the Michaelis-Menten constant for GTP, indicating that behaves like an uncompetitive inhibitor of the GTPase activity favoring the GTP form of FtsZ in the polymers.

Domain folding and flexibility of Escherichia coli FtsZ determined by tryptophan site-directed mutagenesis.

FtsZ has two domains, the amino GTPase domain with a Rossmann fold, and the carboxyl domain that resembles the chorismate mutase fold. Bioinformatics analyses suggest that the interdomain interaction is stronger than the interaction of the protofilament longitudinal interfaces. Crystal B factor analysis of FtsZ and detected conformational changes suggest a connection between these domains.

Fluorescence resonance energy transfer and molecular modeling studies on 4',6-diamidino-2-phenylindole (DAPI) complexes with tubulin.

The goal of this work was to determine the binding properties and location of 4',6-diamidino-2-phenylindole (DAPI) complexed with tubulin. Using fluorescence anisotropy, a dissociation constant of 5.2+/-0.

Tubulin equilibrium unfolding followed by time-resolved fluorescence and fluorescence correlation spectroscopy.

The pathway for the in vitro equilibrium unfolding of the tubulin heterodimer by guanidinium chloride (GdmCl) has been studied using several spectroscopic techniques, specifically circular dichroism (CD), two-photon Fluorescence Correlation Spectroscopy (FCS), and time-resolved fluorescence, including lifetime and dynamic polarization. The results show that tubulin unfolding is characterized by distinct processes that occur in different GdmCl concentration ranges. From 0 to 0.