Detection of an intermediate during unfolding of bacterial cell division protein FtsZ: loss of functional properties precedes the global unfolding of FtsZ.
Using environment-sensitive fluorescence of 1-anilinonaphthalene-8-sulfonic acid, polarization of fluorescein 5'-isothiocyanate-labeled FtsZ, and far-UV circular dichroism spectroscopy, the chemical unfolding of FtsZ was found to proceed through two steps. The first step of the urea-induced unfolding produced an intermediate, which then unfolded at higher concentrations of urea. The intermediate state contains native-like secondary structure and much less tertiary structure compared with the native state. It is distinct from the native state as well as from the unfolded state. Similar to urea-induced unfolding of FtsZ, thermal unfolding of FtsZ also occurs in two steps. The midpoints for the first and second thermal unfolding transitions were found to be 38 +/- 4 and 77 +/- 5 degrees C, respectively. Further, the functional properties of FtsZ are extremely sensitive to urea, guanidium chloride, and sodium dodecyl sulfate. For example, 50% inhibition of the FtsZ assembly and GTP hydrolysis occurred at 0.1 and 0.2 m of urea, respectively. FtsZ lost its functional properties before any significant perturbation in the secondary or tertiary structure was detected by using several fluorescence techniques and far UV-CD indicating preferential local unfolding of the functional region(s). In addition, the unfolded FtsZ regains its ability to polymerize fully upon removal of urea. The data taken together suggest that FtsZ unfolds reversibly through a multistep process, and local responses that inhibit functional properties precede the global transition of FtsZ to the unfolded state.
Santra
Manas K
MK
School of Biosciences and Bioengineering, Indian Institute of Technology, Bombay, Powai, Mumbai 400 076, India.
Panda
Dulal
D
D000817:Anilino Naphthalenesulfonates / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry*; D002942:Circular Dichroism; D003598:Cytoskeletal Proteins; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D005456:Fluorescent Dyes / Q000494:pharmacology; D020558:GTP Phosphohydrolases / Q000378:metabolism; D019791:Guanidine / Q000737:chemistry; D006868:Hydrolysis; D008027:Light; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012542:Scattering, Radiation; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry; D013053:Spectrophotometry; D013501:Surface-Active Agents / Q000494:pharmacology; D013696:Temperature; D013997:Time Factors; D014466:Ultraviolet Rays; D014508:Urea / Q000494:pharmacology
D000817:Anilino Naphthalenesulfonates; D001426:Bacterial Proteins; D003598:Cytoskeletal Proteins; D005456:Fluorescent Dyes; C071525:FtsZ protein, Bacteria; D013501:Surface-Active Agents; D012967:Sodium Dodecyl Sulfate; C027132:1-anilino-8-naphthalenesulfonate; D014508:Urea; D020558:GTP Phosphohydrolases; D019791:Guanidine