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1.
Biochim Biophys Acta ; 1844(7): 1193-200, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24704635

RESUMO

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. Steady-state fluorescence characterization showed the Trp senses different environments and displays low solvent accessibility. Time-resolved fluorescence data indicated that the main conformational changes in FtsZ occur at the interaction surface between the N and C domains, but also minor rearrangements were detected in the bulk of the N domain. Surprisingly, despite its location near the bottom protofilament interface at the C domain, the Trp 275 fluorescence lifetime did not report changes between the GDP and GTP states. The equilibrium unfolding of FtsZ features an intermediate that is stabilized by the nucleotide bound in the N-domain as well as by quaternary protein-protein interactions. In this context, we characterized the unfolding of the Trp mutants using time-resolved fluorescence and phasor plot analysis. A novel picture of the structural transition from the native state in the absence of denaturant, to the solvent-exposed unfolded state is presented. Taken together our results show that conformational changes between the GDP and GTP states of FtsZ, such as those observed in FtsZ unfolding, are restricted to the interaction surface between the N and C domains.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/genética , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Mutação/genética , Triptofano/genética , Proteínas de Bactérias/metabolismo , Dicroísmo Circular , Proteínas do Citoesqueleto/metabolismo , Guanosina Difosfato/química , Guanosina Trifosfato/química , Modelos Moleculares , Mutagênese Sítio-Dirigida , Conformação Proteica , Dobramento de Proteína , Espectrometria de Fluorescência
2.
Biophys J ; 102(9): 2176-85, 2012 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-22824282

RESUMO

FtsZ is a major protein in bacterial cytokinesis that polymerizes into single filaments. A dimer has been proposed to be the nucleating species in FtsZ polymerization. To investigate the influence of the self-assembly of FtsZ on its unfolding pathway, we characterized its oligomerization and unfolding thermodynamics. We studied the assembly using size-exclusion chromatography and fluorescence spectroscopy, and the unfolding using circular dichroism and two-photon fluorescence correlation spectroscopy. The chromatographic analysis demonstrated the presence of monomers, dimers, and tetramers with populations dependent on protein concentration. Dilution experiments using fluorescent conjugates revealed dimer-to-monomer and tetramer-to-dimer dissociation constants in the micromolar range. Measurements of fluorescence lifetimes and rotational correlation times of the conjugates supported the presence of tetramers at high protein concentrations and monomers at low protein concentrations. The unfolding study demonstrated that the three-state unfolding of FtsZ was due to the mainly dimeric state of the protein, and that the monomer unfolds through a two-state mechanism. The monomer-to-dimer equilibrium characterized here (K(d) = 9 µM) indicates a significant fraction (~10%) of stable dimers at the critical concentration for polymerization, supporting a role of the dimeric species in the first steps of FtsZ polymerization.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/ultraestrutura , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/ultraestrutura , Modelos Químicos , Modelos Moleculares , Ureia/química , Dimerização , Polímeros/química , Desnaturação Proteica , Dobramento de Proteína
3.
Biophys Chem ; 151(3): 111-8, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20561743

RESUMO

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. The residue substitutions had little impact on the native conformation. UV-absorption and CD features were identified and ascribed to specific aromatic residues. Time-resolved fluorescence showed that most of the fluorescence decay of ESP tryptophans is due to a discrete exponential component with a lifetime of 5-6ns. Fluorescence polarization measurements indicated that fluorescence of Trp 210 is nearly independent of the fluorescence of Trp 229 and Trp 251, whereas a substantial energy homotransfer between the latter pair takes place. The spectroscopic information was rationalized on the basis of structural considerations and should help in the interpretation and monitoring of the changes at the sub domain level during the conformational transitions and fluctuations of ESP and other Class A beta-lactamases.


Assuntos
Bacillus/enzimologia , Proteínas Mutantes/química , Mutação , Fenômenos Ópticos , Penicilinase/química , Triptofano/metabolismo , Absorção , Biocatálise , Modelos Moleculares , Mutagênese , Proteínas Mutantes/genética , Proteínas Mutantes/isolamento & purificação , Proteínas Mutantes/metabolismo , Penicilinase/genética , Penicilinase/isolamento & purificação , Penicilinase/metabolismo , Dobramento de Proteína , Estrutura Terciária de Proteína , Espectrometria de Fluorescência , Espectrofotometria Ultravioleta
4.
Arch Biochem Biophys ; 465(2): 315-9, 2007 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-17678870

RESUMO

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. The results presented in this work support a cooperative polymerization mechanism in which the binding of DAPI favors protofilament lateral interactions and the stability of the resulting polymers.


Assuntos
Proteínas de Escherichia coli/química , Escherichia coli/metabolismo , GTP Fosfo-Hidrolases/antagonistas & inibidores , Indóis/química , Dimerização , Ativação Enzimática , Complexos Multiproteicos/química , Ligação Proteica
5.
Protein Sci ; 16(8): 1543-56, 2007 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-17656575

RESUMO

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. The unfolding/folding characteristics of each domain of FtsZ were tested by introducing tryptophans into the flexible region of the amino (F135W) and the carboxyl (F275W and I294W) domains. As a control, the mutation F40W was introduced in a more rigid part of the amino domain. These mutants showed a native-like structure with denaturation and renaturation curves similar to wild type. However, the I294W mutant showed a strong loss of functionality, both in vivo and in vitro when compared to the other mutants. The functionality was recovered with the double mutant I294W/F275A, which showed full in vivo complementation with a slight increment of in vitro GTPase activity with respect to the single mutant. The formation of a stabilizing aromatic interaction involving a stacking between the tryptophan introduced at position 294 and phenylalanine 275 could account for these results. Folding/unfolding of these mutants induced by guanidinium chloride was compatible with a mechanism in which both domains within the protein show the same stability during FtsZ denaturation and renaturation, probably because of strong interface interactions.


Assuntos
Proteínas de Escherichia coli/química , Triptofano/química , Sequência de Aminoácidos , Escherichia coli/enzimologia , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , GTP Fosfo-Hidrolases/química , GTP Fosfo-Hidrolases/metabolismo , Guanidina/farmacologia , Guanosina Difosfato/química , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/química , Guanosina Trifosfato/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação Puntual , Dobramento de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , Triptofano/genética
6.
Protein Sci ; 15(3): 410-9, 2006 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-16452620

RESUMO

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+/-0.4 microM for the DAPI-tubulin complex was determined, slightly lower than that for the tubulin S complex. The influence of the C-terminal region on the binding of DAPI to tubulin was also characterized. Using FRET experiments, and assuming a kappa2 value of 2/3, distances between Co2+ bound to its high-affinity binding site and the DAPI-binding site and 2',3'-O-(trinitrophenyl)guanosine 5'-triphosphate bound to the exchangeable nucleotide and the DAPI-binding site were found to be 20+/-2 A and 43+/-2 A, respectively. To locate potential DAPI-binding sites on tubulin, a molecular modeling study was carried out using the tubulin crystal structure and energy minimization calculations. The results from the FRET measurements were used to limit the possible location of DAPI in the tubulin structure. Several candidate binding sites were found and these are discussed in the context of the various properties of bound DAPI.


Assuntos
Corantes Fluorescentes/química , Indóis/química , Modelos Moleculares , Tubulina (Proteína)/química , Animais , Sítios de Ligação , Polarização de Fluorescência , Transferência Ressonante de Energia de Fluorescência , Guanosina Trifosfato/análogos & derivados , Guanosina Trifosfato/química , Fragmentos de Peptídeos/química , Tubulina (Proteína)/metabolismo
7.
Protein Sci ; 13(1): 81-8, 2004 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-14691224

RESUMO

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 0.5 M GdmCl, a slight alteration of the tubulin heterodimer occurs, as evidenced by a small, but reproducible increase in the rotational correlation time of the protein and a sharp decrease in the secondary structure monitored by CD. In the range 0.5-1.5 M GdmCl, significant decreases in the steady-state anisotropy and average lifetime of the intrinsic tryptophan fluorescence occur, as well as a decrease in the rotational correlation time, from 48 to 26 nsec. In the same GdmCl range, the number of protein molecules (labeled with Alexa 488), as determined by two-photon FCS measurements, increases by a factor of two, indicating dissociation of the tubulin dimer into monomers. From 1.5 to 4 M GdmCl, these monomers unfold, as evidenced by the continual decrease in the tryptophan steady-state anisotropy, average lifetime, and rotational correlation time, concomitant with secondary structural changes. These results help to elucidate the unfolding pathway of the tubulin heterodimer and demonstrate the value of FCS measurements in studies on oligomeric protein systems.


Assuntos
Guanidina/farmacologia , Dobramento de Proteína , Espectrometria de Fluorescência , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismo , Animais , Anisotropia , Encéfalo/metabolismo , Galinhas , Dicroísmo Circular , Dimerização , Relação Dose-Resposta a Droga , Fluorescência , Polarização de Fluorescência , Corantes Fluorescentes , Guanidina/química , Hidrazinas , Cinética , Desnaturação Proteica , Estrutura Secundária de Proteína , Rotação , Manejo de Espécimes , Triptofano/química , Tubulina (Proteína)/efeitos dos fármacos , Tubulina (Proteína)/isolamento & purificação
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