Effect of urea denaturation on tryptophan fluorescence and nucleotide binding on tubulin studied by fluorescence and NMR spectroscopic methods.
Tubulin, the major protein of microtubules, has been shown to be an example of protein undergoing multistep unfolding. Local unfolding and stepwise loss of a number of characteristic functions were demonstrated. In order to understand urea induced effects on tryptophan fluorescence and nucleotide binding on tubulin, both fluorescence and NMR techniques were used. Tubulin was denatured by different urea concentrations. The present experiments were carried out at concentrations of tubulin (to approximately 10 microM) at which most of the protein will be in the dimeric state. Quenching studies in the presence of KI suggest that all the tryptophans are fairly solvent exposed. Similar studies using acrylamide as quencher, suggest unfolding of tubulin at these protein concentrations to be an apparent two state process between the native and the completely unfolded states unlike at low concentrations where a partially folded intermediate was observed. No observable effects of the nucleotide or the metal ion on tryptophan fluorescence were observed. An attempt was made using NMR to monitor the changes in the nucleotide interaction with tubulin as the protein is unfolded by urea denaturation. No significant effects were observed in the binding of the nucleotide to tubulin by urea denaturation.
Kuchroo
K
K
Department of Chemical Sciences, Tata Institute of Fundamental Research, Colaba, Mumbai, India.
Maity
H
H
Kasturi
S R
SR
D020106:Acrylamide / Q000737:chemistry; D000818:Animals; D001921:Brain / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D006041:Goats; D006160:Guanosine Triphosphate / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D009711:Nucleotides / Q000378:metabolism*; D011485:Protein Binding; D011489:Protein Denaturation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013997:Time Factors; D014364:Tryptophan / Q000378:metabolism*; D014404:Tubulin / Q000378:metabolism*; D014508:Urea / Q000737:chemistry*
D009711:Nucleotides; D014404:Tubulin; D020106:Acrylamide; D006160:Guanosine Triphosphate; D014364:Tryptophan; D014508:Urea